Lentivirus and Retrovirus 101 basic biology

ShayWright Protocol

Handling Retroviral or Lentiviral Supernatants

1) Before doing TC involving viral supernatants place a small bucket of bleach inside the TC hood (Spray the bucket down with ethanol and wipe it off prior to placing it in the hood)

2) Every pipette tip that comes in contact with viral supernatant should be placed in the bleach bucket and left there for at least 20 minutes Aspirate bleach into the tip to at least the level to which you filled the tip with viral supernatant (If the tip held 10ml of viral supernatant you should aspirate at least 10ml of bleach before removing the tip from the pipette-aid and letting the tip sit in the bleach bucket) The same applies for syringes and filters used with viral supernatant

3) Vials conical tubes cell scrapers or anything else that has touched viral supernatant should be thrown into the bleach bucket and soaked for 20 minutes

4) If you are disposing of the viral supernatant simply pipette the media into the bleach bucket and then bleach your tip as usual

5) TC dishes that held cells that made or were being infected with virus should be filled with bleach and left in the sink for at least 20 minutes (If you had 10ml of media in a 10cm dish put at least 10ml of bleach into the dish)

6) After the 20 minute soak you can dump the bleach into the sink and throw the plastic in the regular biohazard trash for autoclaving

Second vs Third Generation Lentivirus

Second generation viruses use transfection of three plasmids into the packaging cellsYour lentivirus expression vector a VSVG plasmid such as PMD2G illustrated below and a gutted lentivirus expressing gag coding for the virion main structural proteins pol responsible for the retrovirus-specific enzymes and rev which endcodes a posttranscriptional regulator necessary for efficient gag and pol expression In the third generation system the rev is on a separate plasmid and thus requires 4 different plasmids The lentiviruses we got from Geron are third generation almost all the other lentirsquos in the lab are second generationThird generation offers maximal biosafety but is more cumbersome The illustration below are vectors

made by Trono

pCMV-dR874psPAX2 (ldquopsPax2rdquo) and the envelope plasmid pMD2G are available from Addgene (wwwaddgeneorg) psPax2 = plasmid 12260 pMD2G plasmid 12259

Lentiviruses and retrovirus are both large and are partially retained in 022 um filters thus use 045 um filters to remove any contaminating cells

According to the TRC consortium viral harvest growth media containing 10 serum + 11gmL supplemental BSA is equivalent to viral harvest media containing 30 serum ndash both produce viral stocks with similar high titer (2x higher than in 10 serum) The BSA-supplemented media is more cost effective easier to mix in standard 500mL media bottles and may be preferred when transfecting cells that are sensitive to serum

The half-life at 37C for both lenti- and retroviruses is about 4-6 hours so one typically collects supernatants twice a daymdasheg first thing in the morning and last thing at the end of the day

Matt Porteus showed that including caffeine (to block DNA damage signaling and checkpoint activities) during lentiviral production increased titers 2-4-fold 24 or 8 mM caffeine is added the morning after transfection (17 hours) and supernatant collected (in the presence of caffeine each time) at 48 hours and morningsevenings thereafter According to JYrsquos tests 2mM caffeine was the best compromise between toxicity and increased titer He made a 40 mM stock (20x) in Medium X filter sterilized and added it ~12 hours after transfection No tests have been done on retroviral titers

Ecotropic refers to a virus that has the surface env protein that will bind to mouse but not human cells while amphotropic refers to an envelope protein that will bind to both An amphotropic packaging cell line expresses lots of the amphotropic env and is resistant to infection by a retrovirus expressing the amphotropic env since all of its receptors are already tied up by the ligand it is already making Retroviruses sometimes get rearranged following transfection into packaging cells producing some non-infectious particles These can be minimized by first transfecting the retrovirus proviral plasmid into an ecotropic packaging line and then using the supernatant to infect an amphotropic packaging line Only competent particles can infect the amphotropic line and this thus eliminates all of the rearrangements so that one ends up with higher titers

We have both transient and stable systems for the production of retroviral supernatants In general a retrovirus that is for a special purpose and isnrsquot going to be used over and over is most conveniently just packaged in the transient line PhoenixA (A= amphotropic) Routinely used viruses are more conveniently produced in stable packaging lines For this first transfect into the transient packaging line PhoenixE (E=ecotropic) and then take the supernatant and use it to infect the stable amphotropic packaging line PA317 After selection using the appropriate drug the stable line is easily expanded to produce as much supernatant as one wants and can be frozen down and later thawed for additional supernatant production PE501 is the name of our stable ecotropic retroviral packaging line but we rarely use it since the transient PhoenixE takes less time

IMPORTANT retroviral packaging lines are immortal but they LOOSE expression of their packaging factors over time We have reselected for retention of the factors (a mess since diphtheria toxin resistance is one of the selection markers) and designated the reselected cells PD 0 BE SURE to keep track of PDs after thawing the cells and donrsquot keep them in culture for more than 60-90 PDs Refreeze low passage cells after thawing themThe VSVG plasmid expressing the ligand for lentiviral packaging is toxic to cells so stable packaging lines for lentiviral production arenrsquot readily available (some attempts to use an inducible VSVG have been done)Hela cells are somewhat resistant to retroviral infection perhaps because they are infected with HPV The env protein from HPV may be binding the receptor and making it unavailable Several approaches around this include 1) co-transfecting the proviral DNA into Phoenix A along with a VSVG env plasmid (from the lenti systems) VSVG uses a different cellular receptor and thus gets in 2) treating Hela transiently with tunicamycin to block processing of potential endogenous env proteins We actually havenrsquot tried this and someone should at some point based on JVirol 19926678-42 They pretreated CHO cells with 01-03 ugml tunicamycin for 19 hours prior to infection with viral supernatants

Retroviruses only infect dividing cells and a good retroviral supernatant can infect 10-50 of the cells Infecting the cells several times is OK Lentiviruses can integrate into non-dividing cells

Number of viral insertions into genome with gt1 MOI 0 1 2 3 gt3 gt25

0 100 0 0 0 0 001 90 9 0 0 0 0

11 02 82 16 2 0 0 012 03 74 22 3 0 0 018 04 67 27 5 1 0 023 05 61 30 8 1 0 027 06 55 33 10 2 0 032 07 50 35 12 3 1 0

08 45 36 14 4 1 009 41 37 16 5 1 0

1 37 37 18 6 2 03 5 15 22 22 35 0

30 0 0 0 0 100 84

GFP negative GFP+ MOI100 00001 0000190 10 0182 18 0274 26 0367 33 0461 39 0555 45 0650 50 0745 55 0841 59 0937 63 15 95 30 100 30

0

10

20

30

40

50

60

70

01 02 03 04 05 06 07 08 09 1

G

FP+

cells

MOI

MOI vs GFP

and much higher titers can be produced The tables below indicate the relationship between the number of cells that are GFP+ after infection with a GFP-lentivirus and the number of integrations per cell The multiplicity-of-infection (MOI) refers to the number of infectious particles per cell If one wants only a single integrant per cell using an MOI such that lt30 of the cells turn green is the best approach For many situations a high MOI is fine

ShayWright retroviral protocol 2006

Retroviral Protocols

Discard pipets dishes into 50 bleach

Treat retroviral supernatants with respect but not panic the half-life of the virus at 37 degrees is about 4 hours

hTERT is not an oncogenendashit does not confer growth advantages by itself

pBabepuro hTERT-PA317 cells should be resistant to 3 ugml puromycin We do not normally maintain them in puro

Wear gloves etc

To harvest supernatants

Grow packaging cells to near confluence or confluence Replace medium with ldquominimalrdquo amount of fresh medium to adequately cover the dish (eg 12-15 ml for a 15 cm dish with 150 square cm of surface area)

Harvest medium after 6-8 hours or after overnight and replace with fresh medium for the next harvest Harvesting can be repeated twiceday often for 3-4 days until the cell layer detaches or the medium gets acidic too quickly The more confluent the cells the higher the viral titers will be Supernatants should be filtered through a 045 micron sterile filter and can be used fresh or frozen and stored for up to about six months at -80 degrees Freezing causes as loss of about 50 of infectivity but since the infectivity is usually much more than needed for most tissue culture applications it doesnrsquot matter

Convenient method Harvest medium with a 12 ml syringe (no need to use a needle-just shake off the clinging drop back into the dish) and attach the syringe to a 045 micron filter filter it into a 15 ml tube etc

To infect cells

Make viral supernatants 4 microgramsml with polybrene (some cells are sensitive and need 2 ugml polybrene) If needed volumes can be increased (if need to infect multiple dishes or large dishes etc) by diluting the viral supernatant 11 with polybrene-containing fresh medium

Replace medium of growing cells (retroviruses only infect dividing cells thus cells canrsquot be confluent) with enough viral supernatant to cover Leave on at least four hours anything more than 8 hours is probably a waste of time If needed one can infect first thing in the morning then again overnight at the end of the day One infection is usually enough but can do 2-3 times if a cell type proves hard to infect After a 36-48 hour expression period put the cells under selection (puro neo etc)

Sample Retroviral Production Protocolmdashde Lange lab

Required SolutionsPolybreneStock is 10000x in medium stored at 4C (40 mgml) and user stock is 100x Final concentration is 4 μgmlMedium for Phoenix cellsDMEM 10 FBS 1 Non-essential amino acids 1 Pen-Strep 1 Glutamate Store medium at 4C warm to 37C before use25 M CaCl2Filter sterilize and store aliquots at -20C2x HBS50 mM HEPES pH 70510 mM KCl12 mM Dextrose280 mM NaCl15 mM Na2HPO4 (FW 14196)The final pH of the solution should be 705 +- 005 Filter through a 02 μm filter aliquot and store at -20degC Try to avoid multiple freezethaw cycles To thaw warm to room temperature and invert or vortex the tube to achieve uniform mixing Although it is unclear why this occurs the ability of the 2x HBS solution to produce working CaPO4 precipitates deteriorates after 6 months to one year even when the 2x HBS solution is stored at -20degCBleachAll materials that have been in contact with the retrovirus must be bleached thoroughly before discarding in the biohazard waste

Virus productionDay 0 Plate 25 x 106 Phoenix packaging cells in 9 ml medium10 cm dish in the afternoon Note It is very important to have good single cells suspensions (trypsinize well) and to evenly distribute the cellsDay 1 Transfect cells with 20 g DNA (plusmn24 hrs after plating) using CaPO4 precipitation Note At the moment of transfection the cell density should be plusmn 40-50 such that the cells will be about 90 confluent at Day 3 and completely confluent at Day 4 1 In a 2 ml eppendorf tube mix

50 ul of 25 M CaCl220 ug DNA (Qiagen prep purified)MQ up to total of 500 ul2 While vortexing the tube slowly add 500 l 2x HBS drop by dropNote if more of the same transfection mix in 15 ml tube (4 max)3 Add the 1 ml mix drop by drop to the cells in medium and evenly distribute by swirling the plate4 Place cells back in incubatorDay 2 Change medium 5-20 hours after transfection (precipitate is very fine) Late in the afternoon or in evening replace medium with 9 ml of fresh medium Note Phoenix cells detach easily be careful with all medium changesDay 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs after transfection and not later than 24 hrs after changing medium)1 Remove virus-containing medium and set aside for a moment to supply the packaging cells with 9 mls of fresh medium (1-2 plates at a time)2 Filter the virus-containing medium through a 045 um filter and immediately use for infectionDay 4 Collect second supernatant (T2) in the morningCollect third supernatant (T3) in the evening (8 hrs after T2)

Lentiviral Packaging Protocols

Below are a whole series of different protocols Things to consider in usingdesigning your own protocol including caffeine can increase titers 2-4x some protocols claim higher titers in the absence of antibiotics some protocols claim higher titers using 30 serum or 10 serum + 1000 mgml BSA (Note JY claimed higher titers with lowering serum to 5) for large scale production (eg many 15 cm plates for libraries) consider using CaPO4 ppt transfection protocols I donrsquot know if anyone has tested combining caffeine antibiotic free and high protein

Polyjet protocol from Guido Stadler 2011

Transfection of 293 cells with PolyJet (Signagen SL10068B)seed 800k (6 well) 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest gelatin is not needed for routine culture of 293 cells Myoblast group routinely coats plates with gelatin itrsquos easy)change medium to 15 ml fresh growth medium 30-60 min before transfectiondilute 15 ug DNA into 50250 ul medium X vortex gently and spin down

Lentivirus production in 6 well dishes 075 ug target plasmid 075 ug helper plasmids (equimolar ratio of helper plasmids is recommended ~049 ug psPAX2 ~026 ug pMD2G)dilute 315 ul poly jet into 50250 ul medium X vortex gently and spin downadd diluted poly jet to the diluted DNA (not the other way round) vortex and spin down incubate 15 min (not longer than 20) at RTadd the mixture drop-wise onto the medium in the dish mix by swirling the platechange medium 12-18 h after transfection (5 h for sensitive cells)use 2mM caffeine in medium for virus production

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

Lentiviruses and retrovirus are both large and are partially retained in 022 um filters thus use 045 um filters to remove any contaminating cells

According to the TRC consortium viral harvest growth media containing 10 serum + 11gmL supplemental BSA is equivalent to viral harvest media containing 30 serum ndash both produce viral stocks with similar high titer (2x higher than in 10 serum) The BSA-supplemented media is more cost effective easier to mix in standard 500mL media bottles and may be preferred when transfecting cells that are sensitive to serum

The half-life at 37C for both lenti- and retroviruses is about 4-6 hours so one typically collects supernatants twice a daymdasheg first thing in the morning and last thing at the end of the day

Matt Porteus showed that including caffeine (to block DNA damage signaling and checkpoint activities) during lentiviral production increased titers 2-4-fold 24 or 8 mM caffeine is added the morning after transfection (17 hours) and supernatant collected (in the presence of caffeine each time) at 48 hours and morningsevenings thereafter According to JYrsquos tests 2mM caffeine was the best compromise between toxicity and increased titer He made a 40 mM stock (20x) in Medium X filter sterilized and added it ~12 hours after transfection No tests have been done on retroviral titers

Ecotropic refers to a virus that has the surface env protein that will bind to mouse but not human cells while amphotropic refers to an envelope protein that will bind to both An amphotropic packaging cell line expresses lots of the amphotropic env and is resistant to infection by a retrovirus expressing the amphotropic env since all of its receptors are already tied up by the ligand it is already making Retroviruses sometimes get rearranged following transfection into packaging cells producing some non-infectious particles These can be minimized by first transfecting the retrovirus proviral plasmid into an ecotropic packaging line and then using the supernatant to infect an amphotropic packaging line Only competent particles can infect the amphotropic line and this thus eliminates all of the rearrangements so that one ends up with higher titers

We have both transient and stable systems for the production of retroviral supernatants In general a retrovirus that is for a special purpose and isnrsquot going to be used over and over is most conveniently just packaged in the transient line PhoenixA (A= amphotropic) Routinely used viruses are more conveniently produced in stable packaging lines For this first transfect into the transient packaging line PhoenixE (E=ecotropic) and then take the supernatant and use it to infect the stable amphotropic packaging line PA317 After selection using the appropriate drug the stable line is easily expanded to produce as much supernatant as one wants and can be frozen down and later thawed for additional supernatant production PE501 is the name of our stable ecotropic retroviral packaging line but we rarely use it since the transient PhoenixE takes less time

IMPORTANT retroviral packaging lines are immortal but they LOOSE expression of their packaging factors over time We have reselected for retention of the factors (a mess since diphtheria toxin resistance is one of the selection markers) and designated the reselected cells PD 0 BE SURE to keep track of PDs after thawing the cells and donrsquot keep them in culture for more than 60-90 PDs Refreeze low passage cells after thawing themThe VSVG plasmid expressing the ligand for lentiviral packaging is toxic to cells so stable packaging lines for lentiviral production arenrsquot readily available (some attempts to use an inducible VSVG have been done)Hela cells are somewhat resistant to retroviral infection perhaps because they are infected with HPV The env protein from HPV may be binding the receptor and making it unavailable Several approaches around this include 1) co-transfecting the proviral DNA into Phoenix A along with a VSVG env plasmid (from the lenti systems) VSVG uses a different cellular receptor and thus gets in 2) treating Hela transiently with tunicamycin to block processing of potential endogenous env proteins We actually havenrsquot tried this and someone should at some point based on JVirol 19926678-42 They pretreated CHO cells with 01-03 ugml tunicamycin for 19 hours prior to infection with viral supernatants

Retroviruses only infect dividing cells and a good retroviral supernatant can infect 10-50 of the cells Infecting the cells several times is OK Lentiviruses can integrate into non-dividing cells

Number of viral insertions into genome with gt1 MOI 0 1 2 3 gt3 gt25

0 100 0 0 0 0 001 90 9 0 0 0 0

11 02 82 16 2 0 0 012 03 74 22 3 0 0 018 04 67 27 5 1 0 023 05 61 30 8 1 0 027 06 55 33 10 2 0 032 07 50 35 12 3 1 0

08 45 36 14 4 1 009 41 37 16 5 1 0

1 37 37 18 6 2 03 5 15 22 22 35 0

30 0 0 0 0 100 84

GFP negative GFP+ MOI100 00001 0000190 10 0182 18 0274 26 0367 33 0461 39 0555 45 0650 50 0745 55 0841 59 0937 63 15 95 30 100 30

0

10

20

30

40

50

60

70

01 02 03 04 05 06 07 08 09 1

G

FP+

cells

MOI

MOI vs GFP

and much higher titers can be produced The tables below indicate the relationship between the number of cells that are GFP+ after infection with a GFP-lentivirus and the number of integrations per cell The multiplicity-of-infection (MOI) refers to the number of infectious particles per cell If one wants only a single integrant per cell using an MOI such that lt30 of the cells turn green is the best approach For many situations a high MOI is fine

ShayWright retroviral protocol 2006

Retroviral Protocols

Discard pipets dishes into 50 bleach

Treat retroviral supernatants with respect but not panic the half-life of the virus at 37 degrees is about 4 hours

hTERT is not an oncogenendashit does not confer growth advantages by itself

pBabepuro hTERT-PA317 cells should be resistant to 3 ugml puromycin We do not normally maintain them in puro

Wear gloves etc

To harvest supernatants

Grow packaging cells to near confluence or confluence Replace medium with ldquominimalrdquo amount of fresh medium to adequately cover the dish (eg 12-15 ml for a 15 cm dish with 150 square cm of surface area)

Harvest medium after 6-8 hours or after overnight and replace with fresh medium for the next harvest Harvesting can be repeated twiceday often for 3-4 days until the cell layer detaches or the medium gets acidic too quickly The more confluent the cells the higher the viral titers will be Supernatants should be filtered through a 045 micron sterile filter and can be used fresh or frozen and stored for up to about six months at -80 degrees Freezing causes as loss of about 50 of infectivity but since the infectivity is usually much more than needed for most tissue culture applications it doesnrsquot matter

Convenient method Harvest medium with a 12 ml syringe (no need to use a needle-just shake off the clinging drop back into the dish) and attach the syringe to a 045 micron filter filter it into a 15 ml tube etc

To infect cells

Make viral supernatants 4 microgramsml with polybrene (some cells are sensitive and need 2 ugml polybrene) If needed volumes can be increased (if need to infect multiple dishes or large dishes etc) by diluting the viral supernatant 11 with polybrene-containing fresh medium

Replace medium of growing cells (retroviruses only infect dividing cells thus cells canrsquot be confluent) with enough viral supernatant to cover Leave on at least four hours anything more than 8 hours is probably a waste of time If needed one can infect first thing in the morning then again overnight at the end of the day One infection is usually enough but can do 2-3 times if a cell type proves hard to infect After a 36-48 hour expression period put the cells under selection (puro neo etc)

Sample Retroviral Production Protocolmdashde Lange lab

Required SolutionsPolybreneStock is 10000x in medium stored at 4C (40 mgml) and user stock is 100x Final concentration is 4 μgmlMedium for Phoenix cellsDMEM 10 FBS 1 Non-essential amino acids 1 Pen-Strep 1 Glutamate Store medium at 4C warm to 37C before use25 M CaCl2Filter sterilize and store aliquots at -20C2x HBS50 mM HEPES pH 70510 mM KCl12 mM Dextrose280 mM NaCl15 mM Na2HPO4 (FW 14196)The final pH of the solution should be 705 +- 005 Filter through a 02 μm filter aliquot and store at -20degC Try to avoid multiple freezethaw cycles To thaw warm to room temperature and invert or vortex the tube to achieve uniform mixing Although it is unclear why this occurs the ability of the 2x HBS solution to produce working CaPO4 precipitates deteriorates after 6 months to one year even when the 2x HBS solution is stored at -20degCBleachAll materials that have been in contact with the retrovirus must be bleached thoroughly before discarding in the biohazard waste

Virus productionDay 0 Plate 25 x 106 Phoenix packaging cells in 9 ml medium10 cm dish in the afternoon Note It is very important to have good single cells suspensions (trypsinize well) and to evenly distribute the cellsDay 1 Transfect cells with 20 g DNA (plusmn24 hrs after plating) using CaPO4 precipitation Note At the moment of transfection the cell density should be plusmn 40-50 such that the cells will be about 90 confluent at Day 3 and completely confluent at Day 4 1 In a 2 ml eppendorf tube mix

50 ul of 25 M CaCl220 ug DNA (Qiagen prep purified)MQ up to total of 500 ul2 While vortexing the tube slowly add 500 l 2x HBS drop by dropNote if more of the same transfection mix in 15 ml tube (4 max)3 Add the 1 ml mix drop by drop to the cells in medium and evenly distribute by swirling the plate4 Place cells back in incubatorDay 2 Change medium 5-20 hours after transfection (precipitate is very fine) Late in the afternoon or in evening replace medium with 9 ml of fresh medium Note Phoenix cells detach easily be careful with all medium changesDay 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs after transfection and not later than 24 hrs after changing medium)1 Remove virus-containing medium and set aside for a moment to supply the packaging cells with 9 mls of fresh medium (1-2 plates at a time)2 Filter the virus-containing medium through a 045 um filter and immediately use for infectionDay 4 Collect second supernatant (T2) in the morningCollect third supernatant (T3) in the evening (8 hrs after T2)

Lentiviral Packaging Protocols

Below are a whole series of different protocols Things to consider in usingdesigning your own protocol including caffeine can increase titers 2-4x some protocols claim higher titers in the absence of antibiotics some protocols claim higher titers using 30 serum or 10 serum + 1000 mgml BSA (Note JY claimed higher titers with lowering serum to 5) for large scale production (eg many 15 cm plates for libraries) consider using CaPO4 ppt transfection protocols I donrsquot know if anyone has tested combining caffeine antibiotic free and high protein

Polyjet protocol from Guido Stadler 2011

Transfection of 293 cells with PolyJet (Signagen SL10068B)seed 800k (6 well) 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest gelatin is not needed for routine culture of 293 cells Myoblast group routinely coats plates with gelatin itrsquos easy)change medium to 15 ml fresh growth medium 30-60 min before transfectiondilute 15 ug DNA into 50250 ul medium X vortex gently and spin down

Lentivirus production in 6 well dishes 075 ug target plasmid 075 ug helper plasmids (equimolar ratio of helper plasmids is recommended ~049 ug psPAX2 ~026 ug pMD2G)dilute 315 ul poly jet into 50250 ul medium X vortex gently and spin downadd diluted poly jet to the diluted DNA (not the other way round) vortex and spin down incubate 15 min (not longer than 20) at RTadd the mixture drop-wise onto the medium in the dish mix by swirling the platechange medium 12-18 h after transfection (5 h for sensitive cells)use 2mM caffeine in medium for virus production

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

IMPORTANT retroviral packaging lines are immortal but they LOOSE expression of their packaging factors over time We have reselected for retention of the factors (a mess since diphtheria toxin resistance is one of the selection markers) and designated the reselected cells PD 0 BE SURE to keep track of PDs after thawing the cells and donrsquot keep them in culture for more than 60-90 PDs Refreeze low passage cells after thawing themThe VSVG plasmid expressing the ligand for lentiviral packaging is toxic to cells so stable packaging lines for lentiviral production arenrsquot readily available (some attempts to use an inducible VSVG have been done)Hela cells are somewhat resistant to retroviral infection perhaps because they are infected with HPV The env protein from HPV may be binding the receptor and making it unavailable Several approaches around this include 1) co-transfecting the proviral DNA into Phoenix A along with a VSVG env plasmid (from the lenti systems) VSVG uses a different cellular receptor and thus gets in 2) treating Hela transiently with tunicamycin to block processing of potential endogenous env proteins We actually havenrsquot tried this and someone should at some point based on JVirol 19926678-42 They pretreated CHO cells with 01-03 ugml tunicamycin for 19 hours prior to infection with viral supernatants

Retroviruses only infect dividing cells and a good retroviral supernatant can infect 10-50 of the cells Infecting the cells several times is OK Lentiviruses can integrate into non-dividing cells

Number of viral insertions into genome with gt1 MOI 0 1 2 3 gt3 gt25

0 100 0 0 0 0 001 90 9 0 0 0 0

11 02 82 16 2 0 0 012 03 74 22 3 0 0 018 04 67 27 5 1 0 023 05 61 30 8 1 0 027 06 55 33 10 2 0 032 07 50 35 12 3 1 0

08 45 36 14 4 1 009 41 37 16 5 1 0

1 37 37 18 6 2 03 5 15 22 22 35 0

30 0 0 0 0 100 84

GFP negative GFP+ MOI100 00001 0000190 10 0182 18 0274 26 0367 33 0461 39 0555 45 0650 50 0745 55 0841 59 0937 63 15 95 30 100 30

0

10

20

30

40

50

60

70

01 02 03 04 05 06 07 08 09 1

G

FP+

cells

MOI

MOI vs GFP

and much higher titers can be produced The tables below indicate the relationship between the number of cells that are GFP+ after infection with a GFP-lentivirus and the number of integrations per cell The multiplicity-of-infection (MOI) refers to the number of infectious particles per cell If one wants only a single integrant per cell using an MOI such that lt30 of the cells turn green is the best approach For many situations a high MOI is fine

ShayWright retroviral protocol 2006

Retroviral Protocols

Discard pipets dishes into 50 bleach

Treat retroviral supernatants with respect but not panic the half-life of the virus at 37 degrees is about 4 hours

hTERT is not an oncogenendashit does not confer growth advantages by itself

pBabepuro hTERT-PA317 cells should be resistant to 3 ugml puromycin We do not normally maintain them in puro

Wear gloves etc

To harvest supernatants

Grow packaging cells to near confluence or confluence Replace medium with ldquominimalrdquo amount of fresh medium to adequately cover the dish (eg 12-15 ml for a 15 cm dish with 150 square cm of surface area)

Harvest medium after 6-8 hours or after overnight and replace with fresh medium for the next harvest Harvesting can be repeated twiceday often for 3-4 days until the cell layer detaches or the medium gets acidic too quickly The more confluent the cells the higher the viral titers will be Supernatants should be filtered through a 045 micron sterile filter and can be used fresh or frozen and stored for up to about six months at -80 degrees Freezing causes as loss of about 50 of infectivity but since the infectivity is usually much more than needed for most tissue culture applications it doesnrsquot matter

Convenient method Harvest medium with a 12 ml syringe (no need to use a needle-just shake off the clinging drop back into the dish) and attach the syringe to a 045 micron filter filter it into a 15 ml tube etc

To infect cells

Make viral supernatants 4 microgramsml with polybrene (some cells are sensitive and need 2 ugml polybrene) If needed volumes can be increased (if need to infect multiple dishes or large dishes etc) by diluting the viral supernatant 11 with polybrene-containing fresh medium

Replace medium of growing cells (retroviruses only infect dividing cells thus cells canrsquot be confluent) with enough viral supernatant to cover Leave on at least four hours anything more than 8 hours is probably a waste of time If needed one can infect first thing in the morning then again overnight at the end of the day One infection is usually enough but can do 2-3 times if a cell type proves hard to infect After a 36-48 hour expression period put the cells under selection (puro neo etc)

Sample Retroviral Production Protocolmdashde Lange lab

Required SolutionsPolybreneStock is 10000x in medium stored at 4C (40 mgml) and user stock is 100x Final concentration is 4 μgmlMedium for Phoenix cellsDMEM 10 FBS 1 Non-essential amino acids 1 Pen-Strep 1 Glutamate Store medium at 4C warm to 37C before use25 M CaCl2Filter sterilize and store aliquots at -20C2x HBS50 mM HEPES pH 70510 mM KCl12 mM Dextrose280 mM NaCl15 mM Na2HPO4 (FW 14196)The final pH of the solution should be 705 +- 005 Filter through a 02 μm filter aliquot and store at -20degC Try to avoid multiple freezethaw cycles To thaw warm to room temperature and invert or vortex the tube to achieve uniform mixing Although it is unclear why this occurs the ability of the 2x HBS solution to produce working CaPO4 precipitates deteriorates after 6 months to one year even when the 2x HBS solution is stored at -20degCBleachAll materials that have been in contact with the retrovirus must be bleached thoroughly before discarding in the biohazard waste

Virus productionDay 0 Plate 25 x 106 Phoenix packaging cells in 9 ml medium10 cm dish in the afternoon Note It is very important to have good single cells suspensions (trypsinize well) and to evenly distribute the cellsDay 1 Transfect cells with 20 g DNA (plusmn24 hrs after plating) using CaPO4 precipitation Note At the moment of transfection the cell density should be plusmn 40-50 such that the cells will be about 90 confluent at Day 3 and completely confluent at Day 4 1 In a 2 ml eppendorf tube mix

50 ul of 25 M CaCl220 ug DNA (Qiagen prep purified)MQ up to total of 500 ul2 While vortexing the tube slowly add 500 l 2x HBS drop by dropNote if more of the same transfection mix in 15 ml tube (4 max)3 Add the 1 ml mix drop by drop to the cells in medium and evenly distribute by swirling the plate4 Place cells back in incubatorDay 2 Change medium 5-20 hours after transfection (precipitate is very fine) Late in the afternoon or in evening replace medium with 9 ml of fresh medium Note Phoenix cells detach easily be careful with all medium changesDay 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs after transfection and not later than 24 hrs after changing medium)1 Remove virus-containing medium and set aside for a moment to supply the packaging cells with 9 mls of fresh medium (1-2 plates at a time)2 Filter the virus-containing medium through a 045 um filter and immediately use for infectionDay 4 Collect second supernatant (T2) in the morningCollect third supernatant (T3) in the evening (8 hrs after T2)

Lentiviral Packaging Protocols

Below are a whole series of different protocols Things to consider in usingdesigning your own protocol including caffeine can increase titers 2-4x some protocols claim higher titers in the absence of antibiotics some protocols claim higher titers using 30 serum or 10 serum + 1000 mgml BSA (Note JY claimed higher titers with lowering serum to 5) for large scale production (eg many 15 cm plates for libraries) consider using CaPO4 ppt transfection protocols I donrsquot know if anyone has tested combining caffeine antibiotic free and high protein

Polyjet protocol from Guido Stadler 2011

Transfection of 293 cells with PolyJet (Signagen SL10068B)seed 800k (6 well) 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest gelatin is not needed for routine culture of 293 cells Myoblast group routinely coats plates with gelatin itrsquos easy)change medium to 15 ml fresh growth medium 30-60 min before transfectiondilute 15 ug DNA into 50250 ul medium X vortex gently and spin down

Lentivirus production in 6 well dishes 075 ug target plasmid 075 ug helper plasmids (equimolar ratio of helper plasmids is recommended ~049 ug psPAX2 ~026 ug pMD2G)dilute 315 ul poly jet into 50250 ul medium X vortex gently and spin downadd diluted poly jet to the diluted DNA (not the other way round) vortex and spin down incubate 15 min (not longer than 20) at RTadd the mixture drop-wise onto the medium in the dish mix by swirling the platechange medium 12-18 h after transfection (5 h for sensitive cells)use 2mM caffeine in medium for virus production

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

and much higher titers can be produced The tables below indicate the relationship between the number of cells that are GFP+ after infection with a GFP-lentivirus and the number of integrations per cell The multiplicity-of-infection (MOI) refers to the number of infectious particles per cell If one wants only a single integrant per cell using an MOI such that lt30 of the cells turn green is the best approach For many situations a high MOI is fine

ShayWright retroviral protocol 2006

Retroviral Protocols

Discard pipets dishes into 50 bleach

Treat retroviral supernatants with respect but not panic the half-life of the virus at 37 degrees is about 4 hours

hTERT is not an oncogenendashit does not confer growth advantages by itself

pBabepuro hTERT-PA317 cells should be resistant to 3 ugml puromycin We do not normally maintain them in puro

Wear gloves etc

To harvest supernatants

Grow packaging cells to near confluence or confluence Replace medium with ldquominimalrdquo amount of fresh medium to adequately cover the dish (eg 12-15 ml for a 15 cm dish with 150 square cm of surface area)

Harvest medium after 6-8 hours or after overnight and replace with fresh medium for the next harvest Harvesting can be repeated twiceday often for 3-4 days until the cell layer detaches or the medium gets acidic too quickly The more confluent the cells the higher the viral titers will be Supernatants should be filtered through a 045 micron sterile filter and can be used fresh or frozen and stored for up to about six months at -80 degrees Freezing causes as loss of about 50 of infectivity but since the infectivity is usually much more than needed for most tissue culture applications it doesnrsquot matter

Convenient method Harvest medium with a 12 ml syringe (no need to use a needle-just shake off the clinging drop back into the dish) and attach the syringe to a 045 micron filter filter it into a 15 ml tube etc

To infect cells

Make viral supernatants 4 microgramsml with polybrene (some cells are sensitive and need 2 ugml polybrene) If needed volumes can be increased (if need to infect multiple dishes or large dishes etc) by diluting the viral supernatant 11 with polybrene-containing fresh medium

Replace medium of growing cells (retroviruses only infect dividing cells thus cells canrsquot be confluent) with enough viral supernatant to cover Leave on at least four hours anything more than 8 hours is probably a waste of time If needed one can infect first thing in the morning then again overnight at the end of the day One infection is usually enough but can do 2-3 times if a cell type proves hard to infect After a 36-48 hour expression period put the cells under selection (puro neo etc)

Sample Retroviral Production Protocolmdashde Lange lab

Required SolutionsPolybreneStock is 10000x in medium stored at 4C (40 mgml) and user stock is 100x Final concentration is 4 μgmlMedium for Phoenix cellsDMEM 10 FBS 1 Non-essential amino acids 1 Pen-Strep 1 Glutamate Store medium at 4C warm to 37C before use25 M CaCl2Filter sterilize and store aliquots at -20C2x HBS50 mM HEPES pH 70510 mM KCl12 mM Dextrose280 mM NaCl15 mM Na2HPO4 (FW 14196)The final pH of the solution should be 705 +- 005 Filter through a 02 μm filter aliquot and store at -20degC Try to avoid multiple freezethaw cycles To thaw warm to room temperature and invert or vortex the tube to achieve uniform mixing Although it is unclear why this occurs the ability of the 2x HBS solution to produce working CaPO4 precipitates deteriorates after 6 months to one year even when the 2x HBS solution is stored at -20degCBleachAll materials that have been in contact with the retrovirus must be bleached thoroughly before discarding in the biohazard waste

Virus productionDay 0 Plate 25 x 106 Phoenix packaging cells in 9 ml medium10 cm dish in the afternoon Note It is very important to have good single cells suspensions (trypsinize well) and to evenly distribute the cellsDay 1 Transfect cells with 20 g DNA (plusmn24 hrs after plating) using CaPO4 precipitation Note At the moment of transfection the cell density should be plusmn 40-50 such that the cells will be about 90 confluent at Day 3 and completely confluent at Day 4 1 In a 2 ml eppendorf tube mix

50 ul of 25 M CaCl220 ug DNA (Qiagen prep purified)MQ up to total of 500 ul2 While vortexing the tube slowly add 500 l 2x HBS drop by dropNote if more of the same transfection mix in 15 ml tube (4 max)3 Add the 1 ml mix drop by drop to the cells in medium and evenly distribute by swirling the plate4 Place cells back in incubatorDay 2 Change medium 5-20 hours after transfection (precipitate is very fine) Late in the afternoon or in evening replace medium with 9 ml of fresh medium Note Phoenix cells detach easily be careful with all medium changesDay 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs after transfection and not later than 24 hrs after changing medium)1 Remove virus-containing medium and set aside for a moment to supply the packaging cells with 9 mls of fresh medium (1-2 plates at a time)2 Filter the virus-containing medium through a 045 um filter and immediately use for infectionDay 4 Collect second supernatant (T2) in the morningCollect third supernatant (T3) in the evening (8 hrs after T2)

Lentiviral Packaging Protocols

Below are a whole series of different protocols Things to consider in usingdesigning your own protocol including caffeine can increase titers 2-4x some protocols claim higher titers in the absence of antibiotics some protocols claim higher titers using 30 serum or 10 serum + 1000 mgml BSA (Note JY claimed higher titers with lowering serum to 5) for large scale production (eg many 15 cm plates for libraries) consider using CaPO4 ppt transfection protocols I donrsquot know if anyone has tested combining caffeine antibiotic free and high protein

Polyjet protocol from Guido Stadler 2011

Transfection of 293 cells with PolyJet (Signagen SL10068B)seed 800k (6 well) 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest gelatin is not needed for routine culture of 293 cells Myoblast group routinely coats plates with gelatin itrsquos easy)change medium to 15 ml fresh growth medium 30-60 min before transfectiondilute 15 ug DNA into 50250 ul medium X vortex gently and spin down

Lentivirus production in 6 well dishes 075 ug target plasmid 075 ug helper plasmids (equimolar ratio of helper plasmids is recommended ~049 ug psPAX2 ~026 ug pMD2G)dilute 315 ul poly jet into 50250 ul medium X vortex gently and spin downadd diluted poly jet to the diluted DNA (not the other way round) vortex and spin down incubate 15 min (not longer than 20) at RTadd the mixture drop-wise onto the medium in the dish mix by swirling the platechange medium 12-18 h after transfection (5 h for sensitive cells)use 2mM caffeine in medium for virus production

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

To infect cells

Make viral supernatants 4 microgramsml with polybrene (some cells are sensitive and need 2 ugml polybrene) If needed volumes can be increased (if need to infect multiple dishes or large dishes etc) by diluting the viral supernatant 11 with polybrene-containing fresh medium

Replace medium of growing cells (retroviruses only infect dividing cells thus cells canrsquot be confluent) with enough viral supernatant to cover Leave on at least four hours anything more than 8 hours is probably a waste of time If needed one can infect first thing in the morning then again overnight at the end of the day One infection is usually enough but can do 2-3 times if a cell type proves hard to infect After a 36-48 hour expression period put the cells under selection (puro neo etc)

Sample Retroviral Production Protocolmdashde Lange lab

Required SolutionsPolybreneStock is 10000x in medium stored at 4C (40 mgml) and user stock is 100x Final concentration is 4 μgmlMedium for Phoenix cellsDMEM 10 FBS 1 Non-essential amino acids 1 Pen-Strep 1 Glutamate Store medium at 4C warm to 37C before use25 M CaCl2Filter sterilize and store aliquots at -20C2x HBS50 mM HEPES pH 70510 mM KCl12 mM Dextrose280 mM NaCl15 mM Na2HPO4 (FW 14196)The final pH of the solution should be 705 +- 005 Filter through a 02 μm filter aliquot and store at -20degC Try to avoid multiple freezethaw cycles To thaw warm to room temperature and invert or vortex the tube to achieve uniform mixing Although it is unclear why this occurs the ability of the 2x HBS solution to produce working CaPO4 precipitates deteriorates after 6 months to one year even when the 2x HBS solution is stored at -20degCBleachAll materials that have been in contact with the retrovirus must be bleached thoroughly before discarding in the biohazard waste

Virus productionDay 0 Plate 25 x 106 Phoenix packaging cells in 9 ml medium10 cm dish in the afternoon Note It is very important to have good single cells suspensions (trypsinize well) and to evenly distribute the cellsDay 1 Transfect cells with 20 g DNA (plusmn24 hrs after plating) using CaPO4 precipitation Note At the moment of transfection the cell density should be plusmn 40-50 such that the cells will be about 90 confluent at Day 3 and completely confluent at Day 4 1 In a 2 ml eppendorf tube mix

50 ul of 25 M CaCl220 ug DNA (Qiagen prep purified)MQ up to total of 500 ul2 While vortexing the tube slowly add 500 l 2x HBS drop by dropNote if more of the same transfection mix in 15 ml tube (4 max)3 Add the 1 ml mix drop by drop to the cells in medium and evenly distribute by swirling the plate4 Place cells back in incubatorDay 2 Change medium 5-20 hours after transfection (precipitate is very fine) Late in the afternoon or in evening replace medium with 9 ml of fresh medium Note Phoenix cells detach easily be careful with all medium changesDay 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs after transfection and not later than 24 hrs after changing medium)1 Remove virus-containing medium and set aside for a moment to supply the packaging cells with 9 mls of fresh medium (1-2 plates at a time)2 Filter the virus-containing medium through a 045 um filter and immediately use for infectionDay 4 Collect second supernatant (T2) in the morningCollect third supernatant (T3) in the evening (8 hrs after T2)

Lentiviral Packaging Protocols

Below are a whole series of different protocols Things to consider in usingdesigning your own protocol including caffeine can increase titers 2-4x some protocols claim higher titers in the absence of antibiotics some protocols claim higher titers using 30 serum or 10 serum + 1000 mgml BSA (Note JY claimed higher titers with lowering serum to 5) for large scale production (eg many 15 cm plates for libraries) consider using CaPO4 ppt transfection protocols I donrsquot know if anyone has tested combining caffeine antibiotic free and high protein

Polyjet protocol from Guido Stadler 2011

Transfection of 293 cells with PolyJet (Signagen SL10068B)seed 800k (6 well) 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest gelatin is not needed for routine culture of 293 cells Myoblast group routinely coats plates with gelatin itrsquos easy)change medium to 15 ml fresh growth medium 30-60 min before transfectiondilute 15 ug DNA into 50250 ul medium X vortex gently and spin down

Lentivirus production in 6 well dishes 075 ug target plasmid 075 ug helper plasmids (equimolar ratio of helper plasmids is recommended ~049 ug psPAX2 ~026 ug pMD2G)dilute 315 ul poly jet into 50250 ul medium X vortex gently and spin downadd diluted poly jet to the diluted DNA (not the other way round) vortex and spin down incubate 15 min (not longer than 20) at RTadd the mixture drop-wise onto the medium in the dish mix by swirling the platechange medium 12-18 h after transfection (5 h for sensitive cells)use 2mM caffeine in medium for virus production

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

50 ul of 25 M CaCl220 ug DNA (Qiagen prep purified)MQ up to total of 500 ul2 While vortexing the tube slowly add 500 l 2x HBS drop by dropNote if more of the same transfection mix in 15 ml tube (4 max)3 Add the 1 ml mix drop by drop to the cells in medium and evenly distribute by swirling the plate4 Place cells back in incubatorDay 2 Change medium 5-20 hours after transfection (precipitate is very fine) Late in the afternoon or in evening replace medium with 9 ml of fresh medium Note Phoenix cells detach easily be careful with all medium changesDay 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs after transfection and not later than 24 hrs after changing medium)1 Remove virus-containing medium and set aside for a moment to supply the packaging cells with 9 mls of fresh medium (1-2 plates at a time)2 Filter the virus-containing medium through a 045 um filter and immediately use for infectionDay 4 Collect second supernatant (T2) in the morningCollect third supernatant (T3) in the evening (8 hrs after T2)

Lentiviral Packaging Protocols

Below are a whole series of different protocols Things to consider in usingdesigning your own protocol including caffeine can increase titers 2-4x some protocols claim higher titers in the absence of antibiotics some protocols claim higher titers using 30 serum or 10 serum + 1000 mgml BSA (Note JY claimed higher titers with lowering serum to 5) for large scale production (eg many 15 cm plates for libraries) consider using CaPO4 ppt transfection protocols I donrsquot know if anyone has tested combining caffeine antibiotic free and high protein

Polyjet protocol from Guido Stadler 2011

Transfection of 293 cells with PolyJet (Signagen SL10068B)seed 800k (6 well) 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest gelatin is not needed for routine culture of 293 cells Myoblast group routinely coats plates with gelatin itrsquos easy)change medium to 15 ml fresh growth medium 30-60 min before transfectiondilute 15 ug DNA into 50250 ul medium X vortex gently and spin down

Lentivirus production in 6 well dishes 075 ug target plasmid 075 ug helper plasmids (equimolar ratio of helper plasmids is recommended ~049 ug psPAX2 ~026 ug pMD2G)dilute 315 ul poly jet into 50250 ul medium X vortex gently and spin downadd diluted poly jet to the diluted DNA (not the other way round) vortex and spin down incubate 15 min (not longer than 20) at RTadd the mixture drop-wise onto the medium in the dish mix by swirling the platechange medium 12-18 h after transfection (5 h for sensitive cells)use 2mM caffeine in medium for virus production

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

caffeine stock solution dissolve 40mM in medium X filter (MW=19419 gmol 777 gl (3884 mg50ml) = 40mM)Effectene protocol from Ugur Eskiocak~2010

Transient Transfection of 293FT cells using Effectene for virus production (6 well format)Protocol by Ugur Eskiocak1 The day of transfection dilute 1 ug DNA (05 ug vector + 02 ug PMD2G + 03 ug PsPAX2) in 100 ul (total volume) Buffer EC2 Add 8 ul Enhancer and mix by pipetting up and down Incubate at room temperature for 2-5 min3 Add 8 ul Effectene to the DNA-Enhancer mixture Mix by pipetting up and downIncubate the samples for 5-10 min at room temperature to allow transfection-complex formation4 While complex formation takes place wash trypsinize and collect cells in a 15ml centrifuge tube Count the cell numbers5 Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes Mix by pipetting up and down and immediately add the transfection complexes drop-wise onto well6 Plate 2M cellswell on the top of transfection mix in the 6 well7 Incubate the cells with the transfection complexes under their normal growth conditions and collect viral soups 24 48 and 72 hours after transfection

Effectene protocol from Ryan Laranger for Myoblasts ~2010MAKING hCPK4 VIRUS FOR TRANSDUCTION (in 293FT in MediaX 10S)PMD26 = 285ngul PSPAX2 = 345ngul pUE3-lenti-hCDK4 = 250ngullMIX IN A TOTAL Of lug of DNA contained in 100ul of Buffer EC05 ug of~hCDK4 vector (2 ul) 02 ug of PMD2G (1 ul) 03 ug of PsPAX2 (1 ul)2 bull Add 8 ul of Enhancer and mix by pipetting up and downbull Incubate at room temp for 2-5 minutes3bull Add 15 ul of Effectene to the DNA-Enhancer mixturebull Mix by pipetting up and down bull Incubate for 5-10 f inutes at RT 4Wash trypsinize and collect cells in a 15ml falcon tube Count the cell and dilute them to l millionml5 Add 600 ul of growth medium to the tube containing the transfection complexesPipette up and down and immediately add the transfection complexes drop-wise onto wells6bullPlate2 million cells per well on top of the transfection mix (6 well tray)7bull Incubate cells with the transfection complexes and collect viral sups(replace l5ml of media freeze down 750ul and use 750ul to transduce)

Lentiviral Production Protocol adapted from Marcu lab obtained from JY Kim~2009-The day before transfectionI Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm platein either 10 or 25ml X media respectively-The day of transfection

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

1 Check to make sure cells are at least 25 confluent before continuing2 Combine the appropriate amount of plasmid DNAs in a 15ml conical tubeFor 10cm plates5ug DNA3-4ug psPAX2 (structural vector)1-2ug pMDG2 (envelope vector)10ugTOTALFor I5cm platesI5ug DNA75ug psPAX2 (structural vector)75ug pMD2G (envelope vector)30ugTOTAL

(rest of the protocol is for 15cm plates) 3 Add filtered water to a final volume of 878ul )4 Add 122u12M CaCl2 (stock in -20C walk-in freezer) and mix 5 Add 1000ul of2xHBS (stock in -20C walk-in freezer)6 Use a 1 ml pipette tip and an automated pipettor to generate bubbles for l0s at the bottom of the tube and l0s going up and down the mixed solution7 Let mixture sit for 15m8 Remove appropriate amount of media from 293FT cells so that only 18ml is left9 Add all of mixture to the cells gently and evenly Swirl to distribute evenly and put back in incubator OIN-The day after transfection1 Check transfection efficiency for fluorescent marker if possible2 Change to the media for the cell type for which the virus is being produced-The next day 1 Harvest viral supematant and filter through a 045Um filter(can collect viral supernatant up to 3 days after transfection)

2xHBS 50mM HEPES10mM KCl12mM Dextrose280mM NaCl15mM Na2HPO4

Resuspend in 900 ml H2O adjust pH to 704 (with NaOH if using HEPES free acid with HCl if using HEPES sodium salt) Raise volume to 1000 ml and carefully re-adjust final pH to 705 exactly (IMPORTANT pH needs to be very precise at pH 704 ndash 705) Filter through 022 uM and store in aliquots at -20degC (should be good for up to 6 months)

The RNAi Consortium Protocol11807Section II Lentiviral ProductionIntroductionThis section contains protocols for the production of lentivirus stocks from hairpin-pLKO1 plasmids in 6 cm plates Lentiviral production consists of the following steps

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

Day 0 Seed 293T packaging cellsDay 1 (pm) Transfect packaging cells with 3 lentivirus plasmids(hairpin-pLKO1 vector packaging plasmid envelope plasmid)Day 2 (am) 18 hours post-transfection Remove media replace with fresh high-BSA or high-serum mediaDay 3 (am) 24 hours after media change Harvest virus replace with fresh high-BSA or high-serum mediaDay 4 (am) 24 hours after harvest 1 Harvest virus discard packaging cellsThese procedures should be carried out in accordance with biosafety requirements of the host institutionPart 1 Lentiviral Production in 6 cm platesI MaterialsTransfection-quality plasmid DNA for- hairpin-pLKO1 vector (TRC library plasmid ndash see Section I)- 2nd generation packaging plasmid containing gag pol and rev genes (eg pCMV-dR891 or pCMVdR874psPAX2)- envelope plasmid (eg VSV-G expressing plasmid pMD2G) recommended use endotoxin-free plasmid isolation kits (Qiagen)TransIT-LT1 transfection reagent (Mirus Bio MIR 230056)alternative FuGENE 6 (Roche 1 814 443 or 1 988 387)OPTI-MEM serum-free media (Invitrogen 31985-070)293T packaging cells (recommended passage number lt 10)Cell seeding media Low-antibiotic 293T growth media (DMEM + 10 iFBS + 01x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)05 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)Viral harvest media High-BSA 293T growth media (DMEM + 10 iFBS + 11g100mL BSA + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)50 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)32 mL 20g100mL BSA stock (microbiology-grade Bovine Serum Albumin VWR 14230-738)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)alternative viral harvest media High-serum 293T growth media (DMEM + 30 iFBS + 1x PenStrep)500 mL DMEM (Dulbeccos Modification of Eagles Medium eg Mediatech 10-013-CV)200 mL iFBS (heat-inactivated Fetal Bovine Serum eg HyClone SH3007103)5 mL 100x PenStrep (10000 IUmL penicillin 10000 μgmL streptomycin eg Mediatech 30-002-CI)6 cm tissue culture platesPolypropylene storage tubesII Instructions1 Seed 293T packaging cells at 13-15x105 cellsmL (6 mL per plate) in low-antibiotic growth media(DMEM + 10 iFBS + 01x PenStrep) in 6 cm tissue culture plates2 Incubate cells for 24 hours (37 degC 5 CO2) or until the following afternoon After ~24 hours the cellsshould be ~70 confluent3 Transfect packaging cellsa Prepare a mixture of the 3 transfection plasmidsReagent per 6 cm platepackaging plasmid (eg pCMV-dR891 or pCMV-R874psPAX2) 900 ngenvelope plasmid (eg VSV-GpMD2G) 100 nghairpin-pLKO1 vector 1 μg

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

OPTI-MEM to total volume 10 to 30 μL The volume of OPTI-MEM per well can be adjusted for optimal handlingb Dilute TransIT-LT1 transfection reagent in OPTI-MEM Add the TransIT-LT1 reagent dropwiseand mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing)Incubate 5 minutes at room temperatureReagent per 6 cm plateTransIT-LT1 6 μLOPTI-MEM to total volume 90 μLc Add the 3 plasmid mix dropwise to the diluted TransIT-LT1 reagent and mix by swirling the tip orgently flicking the tubed Incubate the transfection mix for 20 - 30 minutes at room temperaturee Carefully transfer the transfection mix to the packaging cells (in low-antibiotic growth media)The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from theplate The total volume of transfection mix should be 100 to 125 μL per plate4 Incubate cells for 18 hours (37 degC 5 CO2) or until the following morning5 Change media to remove the transfection reagent and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests6 Incubate cells for 24 hours (37 degC 5 CO2)7 Harvest media containing lentivirus at ~40 hours post-transfection Transfer media to a polypropylenestorage tube Replace with 6 mL high-BSA growth media or high serum growth media for viral harvests8 Repeat viral harvesting every 12-24 hours and replace with 6 mL high-BSA growth media or high serum growth media for viral harvests Viral titer tends to decrease in later harvests we typically collect a total of 2-3 time points After the final harvest discard the packaging cells The viral harvests may be pooled as desired9 Spin the media containing virus at 1250 rpm for 5 minutes to pellet any packaging cells that were collected during harvesting Transfer the supernatant to a sterile polypropylene storage tube10 Virus may be stored at 4 degC for short periods (hours to days) but should be frozen at -20 degC or -80 degC for long-term storage To reduce the number of freezethaw cycles aliquot large-scale virus preps to smaller storage tubes prior to long-term storage

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

RESOURCES

Openbiosystems 62K shRNA lentiviral library in pGIPz

MGC 16K cDNA cloneset in pSport6 (a gateway compatible vector) and its lentiviral gateway transfer recipient 711 Map pSSI 14207 pDONR LTR-CMV-attP1-ccdB-CAT-attP2-SV40-Hygro-LTR

pINDUCER lentivirus elicits inducible cDNA and shRNA expression

Meerbrey K L et al PNAS 20111083665-3670

copy2011 by National Academy of Sciences

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

Summary of Gateway system wrt transferring MGC library

The MGC library is an Expression Clone (ie it has attB sites) and thus can only be transferred to what is called a Donor Vector containing attP sites This donor designation is just semantic according to their nomenclaturemdashit could be a retrolenti proviral plasmid with the attP-toxic ccdB-attP sites following a CMV promoter

(NbBsp1407 and BsrGI cute tgtaca at position 9 in attP1amp2Rsa1 is gtac)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

The CcdB protein interferes with E coli DNA gyrase (Bernard and Couturier 1992) thereby inhibiting growth of most E coli strains (eg OmniMAXtrade 2-T1R DH51048644trade TOP10) Because of the lethal effects of the CcdB protein all Gatewayreg vectors containingthe ccdB gene must be propagated in an E coli strain that is resistant to CcdB effects We recommend using the ccdB Survival T1R E coli strain which is resistant to CcdB effects (Bernard and Couturier 1992 Bernard et al 1993 Miki et al 1992) One Shotreg ccdB Survival T1R Chemically Competent E coli are available fromInvitrogen (Catalog no C7510-03) for transformation See page 18 for the genotypeof ccdB Survival T1R E coli

The recombination can occur between DNAs of any topology (ie supercoiled linear or relaxed) although efficiency varies For optimal efficiency perform the BP recombination reaction using10486441048644 Linear attB substrates (see the next page for guidelines to linearize attB expression clones

10486441048644 Supercoiled attP-containing donor vectorNote Supercoiled or relaxed attB substrates may be used but will react less efficientlythan linear attB substrates

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

(thus nomenclature is BP clonase to recombine between B and P sites LR clonase to recombine between L and R sites)

For your convenience we suggest using the following nomenclature to catalog your Gatewayreg vectors and clones

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

(thus CMVpSport6 would be pEXPCMVpSprot6 and need a pDONR-type as recipient)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

Below is the map of the lenti backbone we made so that one can directly transfer MGC clones to a lentivirus The map automatically labeled the att sites as multiple forms but this vector has only attP1 and attP2 sites Use BP Clonase (rather than LR Clonase) to do the transfer

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)

Protocol for inducible cDNA expression gateway cloning from Lu Zhang 2011

1 Design primers flanking cDNA ORF sequence with attB sequence at the ends

2 Perform PCR

(If using the cDNA in pCMV-sports6 vector donrsquot do PCR First linearize the vector with a restriction enzyme that cuts outside the attB sites but not within your cDNA Streak the cut DNA on amp plates to verify adequate cutting and the lack of colonies Use 50ng for BP reaction)

3 Incubate attB-flanked PCR product(50fmol) with donor vector(pDONRtrade221 Kan150ng) overnight using BP Clonase protocol treat with proteinase K for 10min and do transformation

4 Pick colonies and sequence(typically 80 correct)5 Incubate entry clone(150ng or double or more) with destination vector (pINDUCER20 or

21 150ng) for 1 hour using suggested amount on LR Clonase protocol treat with proteinase K for 10 min and do transformation

6 Pick colonies and sequence(about 50 are correct)