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ViraPowerTM Lentiviral Expression Systems TABLE OF CONTENTS PRODUCT DESCRIPTION SHIPPING CONDITIONS STORAGE CONDITIONS STABILITY QC SPECIFICATIONS PROTOCOL & APPLICATION NOTES

Vector elements CMV promoter Use of a promoter other than CMV RARE element VSV-G 3’ polyA SV40 polyA beta-Globin intron RRE (rev responsive element) GP 160 Envelope Protein

Specific Vector details General Cloning Considerations pLenti6/TR pLenti4/TO-V5 pLenti6.2-GW/EmGFP

Virus and vector biology Lentiviral genome content in pLenti vectors Toxicity Multiple Integrants Nuclear Import Lentivirus vs. Adeno-Associated Virus (AAV) Physical Characteristics (based on HIV)

293FT Producer Cell Line General Information Culturing 293FT cells Supplementing 293FT culture medium with Sodium Pyruvate

Experimental Procedures Overview Cloning Transformation

Transformation with Stbl3 E.coli Transformation with One Shot ccdB Survival T1 Phage-Resistant Cells

Transfection Pre-testing of expression vector before transfection for virus production Transfection of 293FT cells

Virus production and harvest Concentration of Virus Titering viral supernatant Polybrene

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Tips to Improve Viral Titers Packaging Limits

Transduction Difference between Lentiviral and Adenoviral transduction FACS analysis with GFP-lenti transduced cells Transduction of virus in vivo and in tissue slices Stable integration of lentiviral constructs Creation of a Double Stable Cell Line Transient transduction Leakiness of expression B Lymphocytes T lymphocytes Transduction of Suspension Cells Monocytes and Macrophages Transient transduction Transduction of Neurnos Embryo transduction Transduction in Chickens Optimizing Lentiviral Transductions

Troubleshooting for Protein Expression Virus rescue

Safety Issues Key Safety Features of the ViraPower™ Lentiviral Expression System gp160 Envelope Protein Recombination or otherwise activation of endogenous retroviruses Activation of oncogenes Rev: splicing and safety in the ViraPower™ Lentiviral system Splicing and safety in the wild-type virus Risk Assessment Explanation of Biosafety Levels 1, 2, 3, 4 (BSL-1, -2, -3, -4)

ALTERNATE PRODUCTS & COMPATIBILITY PRODUCT DOCUMENTATION REFERENCES PRODUCT NAME AND CATALOG NUMBERS COMPONENTS ASSOCIATED PRODUCTS

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PRODUCT DESCRIPTION (back to Table of Content) The ViraPower™ Lentiviral Expression System allows one to create replication-incompetent lentiviruses to deliver and express a gene of interest. The pLenti vector containing a gene of interest is co-transfected into 293FT cells with packaging mix in order to generate virus. Special Features

The virus is loosely based on the HIV-1 strain NL4-3. Can transduce dividing and non-dividing mammalian cells (in culture or in vivo). Invitrogen’s Directional TOPO and Gateway Lentiviral vectors are based on the pRRL vector from Cell

Genesys. Vectors can theoretically accommodate up to ~6kb of inserted foreign gene. Three supercoiled packaging plasmids (gag/pol, rev and VSV-G envelope) are provided to supply helper

functions and viral proteins in trans. Producer cell line (293FT) is provided that will facilitate production of high titer virus. Multiple built-in safety features. Vectors lack gag/pol, envelope and any accessory genes. Gag/pol are supplied in trans on a separate vector ( pLP1). U3 region in the 3'LTR is deleted so that the integrated virus has no promoter elements in the 5' LTR.

Thus, the viruses self-inactivate once they integrate into the host chromosome pLenti6.2 dest vectors are considered a second generation because the SV40 promoter is replaced with the

phosphoglycerate kinase (PGK) promoter. REASON: PGK promoter is resistant to silencing and shows long-term, persistent expression in stem cells. Please remember that the SV40 promoter-driven Bsd selection marker performs poorly in some primary and stem cells. Therefore, it was replaced by the PGK promoter.

The EmGFP lentiviral expression vector is for convenient visual detection of transfection and transduction efficiency. REASON: EmGFP is brighter than cycle3-GFP, as bright as EGFP, and detectable with a standard FITC filter.

N- and C-terminal Lumio fusion lentiviral vectors allow convenient detection and localiztion of proteins in live cells.

Applications

Creating stable cell lines Expressing genes in primary cells Working with neurons or hard to transfect cell types Inducible lentiviral system Needing tissue-specific promoter – can insert promoter of interest RNAi applications Making transgenic animals Animal experiments that require localized gene delivery Target validation Gene library delivery Detection and localization of proteins in live cells, see above under EmGFP and Lumio, both available

since July 15, 2005 SHIPPING CONDITIONS (back to Table of Content)

ViraPower™ Packaging Mix Blue Ice pLenti6/ V5-D-TOPO Reagents Dry Ice pLenti-DEST Gateway vectors Blue Ice pLenti6.2-GW/EmGFP RT pLenti6.2/V5-DEST Gateway Vector and lacZ control

Dry Ice

pLenti6/BLOCK-i-DEST RT

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Gateway Vector Kit pLenti4/BLOCK-iT-DEST Gateway® Vector Kit

RT

pLenti6.2/N & C-Lumio™/V5-DEST and lacZ control

Dry Ice

Lipofectamine 2000 Blue Ice Blasticidin Blue Ice Zeocin RT/Blue Ice 293FT Cell Line Dry Ice One Shot Stbl3 Competent Cells Dry Ice Lumio™ Green In-CellLabeling Kit Blue Ice

STORAGE CONDITIONS (back to Table of Content)

Do not store the vector at -80oC, as too many freeze thaws from -80oC will affect the vector. It is better to store the vector at -20oC, as the vector will not freeze at this temp.

Lentivirus should be stored at –80oC, further comments about virus storage, please see in“ Concentration of virus”.

Virus should be stable at 4oC for overnight, but longer time is not recommended. The ViraPower™ Packing mix should be stored at –20oC. Store Lipofectamine 2000, zeocin, and blasticidin at 4oC. All competent cells should be stored at –80oC Lumio™ Green In-Cell Labeling Kit in the Lumio Lentiviral expression kit is stored at –20oC

STABILITY (back to Table of Content) All reagents are guaranteed stable for 6 months when properly stored. QC SPECIFICATIONS (back to Table of Content)

The pLenti6/V5-D-TOPO vector is lot-qualified using the control reagents included in the kit. Under conditions described in the manual, a 750 bp control PCR product is amplified using a forward primer containing CACC at its 5 end and a reverse primer. The PCR product is TOPO Cloned into the pLenti6/V5-D-TOPO vector and transformed into the One Shot TOP10 chemically competent E. coli included with the kit. Each lot of vector should yield greater than 85% cloning efficiency. Forty transformants are characterized using directional PCR. Of the transformants characterized, greater than 90% should contain an insert in the correct orientation.

The 293FT cells were derived from LTI's 293F cell line, which was screened for BVDV, reovirus, and cytopathic agents. The 293FTs were not similarly screened

Every lot of 293FT cells is tested for mycoplasma. PROTOCOL AND APPLICATION NOTES (back to Table of Content)

Vector elements CMV promoter Use of a promoter other than CMV RARE element VSV-G 3’ polyA SV40 polyA beta-Globin intron

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RRE (rev responsive element) GP 160 Envelope Protein

Specific Vector details General Cloning Considerations pLenti6/TR pLenti4/TO-V5 pLenti6.2-GW/EmGFP

Virus and vector biology Lentiviral genome content in pLenti vectors Toxicity Multiple Integrants Nuclear Import Lentivirus vs. Adeno-Associated Virus (AAV)

293FT Producer Cell Line General Information Culturing 293FT cells Supplementing 293FT culture medium with Sodium Pyruvate

Experimental Procedures Overview Cloning Transformation

Transformation with Stbl3 E.coli Transformation with One Shot ccdB Survival T1 Phage-Resistant Cells

Transfection Pre-testing of expression vector before transfection for virus production Transfection of 293FT cells

Virus production and harvest Titering viral supernatant Concentration of Virus Polybrene Tips to Improve Viral Titers Packaging Limits

Transduction Difference between Lentiviral and Adenoviral transduction Transduction of virus in vivo and in tissue slices Stable integration of lentiviral constructs Creation of a Double Stable Cell Line Transient transduction Leakiness of expression B Lymphocytes T lymphocytes Transduction of Suspension Cells Monocytes and Macrophages Transduction of Neurons Embryo transduction Transduction in Chickens Optimizing Lentiviral Transductions

Troubleshooting for Protein Expression Virus rescue

Safety Issues Key Safety Features of the ViraPower™ Lentiviral Expression System gp160 Envelope Protein Recombination or otherwise activation of endogenous retroviruses Activation of oncogenes Rev: splicing and safety in the ViraPower™ Lentiviral system Splicing and safety in the wild-type virus

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Risk Assessment Explanation of Biosafety Levels 1, 2, 3, 4 (BSL-1, -2, -3, -4)

Vector elements (back to Table of Content) (back to Protocol and Application Notes)

CMV promoter Use of a promoter other than CMV RARE element VSV-G 3’ polyA SV40 polyA beta-Globin intron RRE (rev responsive element) GP 160 Envelope Protein

CMV promoter (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

It can be easily removed (i.e. there are unique restriction sites on both sides). For pLenti6/V5-D-TOPO, ClaI (position 1796) and BamHI (in MCS) can be used.

It will be down regulated in NIH3T3 and often in other fibroblast-type rodent cells Use of a Promoter Other Than CMV (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

The RNAi lentiviral expression vector pLenti6-BLOCK-iT-DEST or the Promoterless Lentiviral Gateway vector pLenti6/R4R2/V5-DEST can be used.

Another option is to insert the sequence of interest within the U3 region of the 3' LTR. This has the advantage that upon reverse transcription and integration each cell will have at least two copies of the cloned elements integrated into the cell (ie. each proviral LTR will supply a copy of the sequence of interest). Insertion into the U3 region of the 3'LTR will not inhibit packaging since the psi-packaging signal is downstream of the transcription start site. However the att sites (for pLenti6/V5-DEST only) will have to be retained or else there will be no integration after packaging and transduction.

Cloning upstream of the CMV promoter will also work, particularly if no more than one copy per cell is needed; the ClaI site can be used.

The Ubc and EF1 promoters are good choices for lentiviral transduction of B and T cells, but we have not tested them in-house. So far only human 293 cells and rat neural stem cells have been tested in-house with lentiviral constructs containing an EF promoter. However, we recommend doing literature searches to see if there's any data on how well EF1a or Ubc promoters work in B and T cells.

RARE element (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

The Retinoic Acid Response Element (RARE) in HIV-1 5' LTR is deleted in Invitrogens lentiviral vectors. This deletion is a built-in safety feature. Due to this deletion, after reverse transcription and integration there will be no promoter elements in the 5' LTR to drive full-length vector transcription. This eliminates any chance of producing infectious virus from a viral-encoded cis element after integration.

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The self-inactivation feature involves deletion of nucleotides -418 to -18 in the U3 LTR region; this deleted region contains the HIV-1 RARE (-349 to -327) among the other promoter elements in the LTR.

VSV-G (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

Theodore Friedmann was the pioneer in the field of lentiviral host range and has a tremendous number of publications and reviews describing VSV-G pseudotyping of retroviruses and their tropisms.

VSV-G binding to target cells is receptor-independent. It interacts with a common phospholipid (phosphatidylserine has been proposed to be the most probable

target) in the target cell membrane and this is how it confers such broad tropism: Mammalian cells, fish, etc (Burns et al Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037).

One group has shown that VSV-G pseudotyped retroviruses can transduce amoebas (see Que et. al. Mol. Biochem. Parasitol. (1999) 99:237-45).

Publications describing the use of lentivirus in insect cells are not available, but that doesn't mean it won't work.

The origin: The San Juan strain of the Indiana serotype (Rose and Gallione, J. Virol. 39:519, 1981). As referenced by Ory et al, PNAS, 93:11400, 1996.

3’ polyA (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

There is no separate polyA signal sequence located anywhere in the Lentiviral genome because that would kill nearly all virus production; the virus uses the 3’ LTR.

The polyA signal in the 3' LTR is AATAAA and is located in the "R" region of the 3'LTR at nucleotides 5335-5340 in pLenti6/V5-DEST. It is not annotated as a feature on the map.

The 3'LTR naturally contains a very strong polyA that is used by all of the transcription cassettes: All of the transcripts coming from the 5'LTR, the CMV promoter AND the SV40 promoter all use the 3'LTR polyA.

Normally, the viral genome that is destined for packaging is produced by transcription starting at the 5'LTR (contains a very strong promoter) and terminates at the 3'LTR. If there was a polyA before the 3'LTR there would be no viral RNA genome for packaging and therefore no functional virus would be produced.

Since the presence of a polyA signal between the LTR sites will cause premature termination of the viral RNA, it is NOT recommended to clone any Invitrogen Superscript II premade library (such as those based on Vector: pCMV-SPORT6) or any clone that has been isolated from such a library into any pLenti expression vectors. Inserts cloned into lentiviral vectors should not have a polyA signal. During construction of a cDNA library using Oligo dT, the native polyA signal (AATAAA or something similar) will be amplified and become part of the cDNA library or its clones. The lentivirus being an RNA virus, during the synthesis of the RNA genome to be packaged, if there is a polyadenylation (polyA) signal in the insert, the RNA will be terminated prematurely. There may be some read through, but the titer will be definitively lower. There is an SV40polyA signal in the vector but it is after the second 3’-LTR (see next section for further information). In order to circumvent premature termination of the lentiviral RNA, one should do the following: The desired gene should first be isolated from the library, cloned into an entry vector such as pEntr/D-TOPO without the polyA signal (i.e. from ATG to Stop), and then transferred into the lentiviral vector. In order to establish a lentiviral expression library, choose a library that was amplified using random hexamers rather than an oligo dT, since such a library would be less likely to include a polyA signal in the insert. Size is not usually a problem - the insert size limit of Lenti is ~5-6kb (Average insert size of the Superscript II premade libraries is ~1.5 – 2.0 kb).

SV40 polyA

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(back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

The SV40 polyA downstream of the 3'LTR in all lentiviral vectors is left over from other cloning steps, is never packaged (since it's beyond the 3'LTR), and is thought only to be somewhat useful during virus production in the 293FT cells. The presence of this SV40 poly A is not absolutely critical in the vector, since the 3' LTR poly A is already present but it should help to reduce the risk of transcriptional interference.

Taken together, the pLenti vectors contain two poly As; one located within the 3' LTR that is derived from HIV-1 and the other, the SV40 poly A just downstream of the 3’LTR. The reason for having both poly As is to reduce the chance of transcriptional interference (for instance, if there was a significant amount of transcriptional read-through that continued through the RSV promoter region, this could potentially interfere with transcription from the RSV promoter, which is critical for production of the viral RNA). Once the lentivirus has integrated in the target cells, the SV40 poly A will not be present (since the virus just extends from the 5' to 3' LTR), but the poly A within the 3' LTR region will still be present and functional. So, all subsequent gene expression relies on the polyA in the R region of the 3'LTR.

beta-Globin intron (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

pLP1 enhances the expression of gag/pol in the producer 293FT cells. It does so by increasing the amount of processed mRNA being exported from the nucleus and does not affect transcription at all.

RRE (rev responsive element) (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

Enhances nuclear export in the presence of rev protein. GP160 Envelope Protein (back to Table of Content) (back to Protocol and Application Notes) (back to Vector elements)

There is a region of overlap after alignment of the gp160 DNA sequence with pLenti6/V5-D-TOPO; however this overlap region does not encode gp160. The first 400 bases of gp160 are deleted in this region. The region of envelope left in the vector lacks an initiator ATG as well as the first 133 amino acids. Any transcript that is produced in the packaging transfection will still lack a functional envelope.

Since the lentiviral vector has a self-inactivating feature, there is a minimal chance of having promoter driven expression of the truncated env fragment left in the vector. The SIN feature ensures that the enhancer region in the 3' LTR does not get copied into the 5' LTR to drive lentiviral genome expression. Even if the virus integrates into a transcriptional hot spot, the 400 bp deletion in the envelope gene assures that functional env doesn't get expressed. The env-deleted region is also upstream of the CMV promoter so the internal promoter does not drive expression of the deleted region.

A portion of gp160 is left in the vector to provide functionality and safety. The part of the envelope region left in the vector comprises the RRE feature. This region ensures that the viral genome gets transported from the nucleus to the cytoplasm to be packaged into virions. Thus, this region of env must be in the transfer vector. The RRE is functional in the presence of the Rev protein. The safety feature here resides in the fact that Rev is provided on another plasmid, pLP2, to minimize against recombination.

Since there is no Gag, pol, rev or functional env genes present in the lentiviral vector, these proteins are not expressed in the target cell.

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Specific Vector details (back to Table of Content) (back to Protocol and Application Notes)

General Cloning Considerations pLenti6/TR pLenti4/TO-V5 pLenti6.2-GW/EmGFP

The pLenti vectors will work as expression vectors by themselves and can be stably selected with blasticidin. Please note though that the vector will be about twice the size of most regular vectors. Therefore you may need to increase the amount of transfected vector to approximate molar equivalents. General Cloning Considerations (back to Table of Content) (back to Protocol and Application Notes) (back to Specific Vector details)

If a pLenti vector is re-engineered to contain an expression cassette in the reverse orientation with respect to the LTRs, the transcripts arising from the promoter in the reverse orientation will interfere with the viral RNA and hence cause a low titer. The HIV-1 genome consists of two identical copies of single-stranded RNA. Generating dsRNA, as could happen in this instance, will reduce titers because the dsRNA will interfere with genome packaging. Also reversing the orientation of the expression cassette with respect to the LTRs will decrease virus titers (Mautino et al, Human Gene Therapy, 11:895, 2000).

pLenti6/TR (back to Table of Content) (back to Protocol and Application Notes) (back to Specific Vector details)

The pLenti6/TR vector encodes a gene of 648 nucleotides (postions 3208-3855), 620 nucleotides coming from the TetR gene (positions 3208-3827) and the extra C-terminal 28 nucleotides (positions 3827-3855; stop codon is included) coming from the cloning. These additional nucleotides are not essential for repression or for tetracycline response.

The pcDNA6/TR vector encodes a gene of 657 nucleotides (positions 1684-2340), 620 nucleotides corresponding to the TetR gene (positions 1684-2303) and the extra C-terminal 37 nucleotides (positions 2304 - 2340; stop codon is included) coming from the cloningThese additional nucleotides were also probably present in the original vectors and are not essential for repression or for tetracycline response.

When the gene sequence encoded by pcDNA6/TR is compared to that encoded by plenti6/TR, they are identical except for an additional 9 nucleotides (AGATCTTAT which encode 3 amino acids Arg-Ser-Tyr) in the C-terminal region of pcDNA6/TR that arose from cloning and are not essential for repression or for tetracycline response. Note that the stop codon that follows Arg-Ser-Tyr in pcDNA6/TR is TAA; the stop codon in pLenti6/TR is TAG.

pLenti4/TO-V5 (back to Table of Content) (back to Protocol and Application Notes) (back to Specific Vector details)

It is possible to use one of the existing pre-made T-REx cell lines and transduce with pLenti4/TO V5 virus containing the gene of interest. However, it is important to test different MOIs of the Lenti4/TO virus, in order to achieve optimal tetracycline regulation.

pLenti6.2-GW/EmGFP (back to Table of Content) (back to Protocol and Application Notes)

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(back to Specific Vector details)

The EmGFP is 719bp and is flanked by attB sites and thus can be moved to a DEST vector via a pDONR vector. Note this vector does not allow one to fuse the EmGFP with a gene of interest. This lentiviral based vector serves as a control vector to test the lentiviral system and allows for optimization of transfection of target cells.

Virus and vector biology (back to Table of Content) (back to Protocol and Application Notes)

Lentiviral genome content in pLenti vectors Toxicity Multiple Integrants Nuclear Import Lentivirus vs. Adeno-Associated Virus (AAV) Physical Characteristics (based on HIV)

Lentiviral genome content in pLenti vectors (back to Table of Content) (back to Protocol and Application Notes) (back to Virus and vector biology)

Very little of the original viral genome is present in Invitrogen’s lentiviral vectors. Invitrogens lentiviral transfer vector has approximately 2.0 kb of viral DNA. Since the wild type virus is

9.7 kb, approximately 20% of the viral DNA is in the transfer vector. This is the part of the viral genome that gets integrated into the target cell together with the gene of interest. Majority of the rest of the viral genome is in the packaging vectors that do not get packaged or integrated into the target cell.

The entire region from the beginning of the 5' LTR to the end of 3' LTR in pLenti6/V5 is 3865 bp. Of this, only 2Kb comes from HIV-1; the remaining nts come from the CMV promoter; Tags + 3Stops; SV40p+EM7+Blasticidin gene. In other words: approx. 80% of the viral genome is deleted from our pLenti vectors

Toxicity (back to Table of Content) (back to Protocol and Application Notes) (back to Virus and vector biology) Invitrogen’s Lentiviral vectors do not carry or express any viral genes and therefore have no associated toxicity issues. Sindbis and SFV vectors express some viral genes in addition to your gene of interest. This is the source of their toxicity. For additional information, see the section on Safety issues. Multiple Integrants (back to Table of Content) (back to Protocol and Application Notes) (back to Virus and vector biology) There is a linear relationship between MOI and integrated copy number. While quantitative data is not available from in house experiments, there is a lot of information in the literature that indicates that as the MOI is increased the number of integrated copies correspondingly increases. Nuclear Import (back to Table of Content) (back to Protocol and Application Notes) (back to Virus and vector biology)

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One of the advantages of lentivirus over other retroviruses is that lentivirus infects the majority of non-

dividing and terminally differentiated cell types (although there are apparently a few rare cell types that may need to go from G0 to at least G1b in order to get completion of the lentiviral reverse transcription and integration process). Lentivirus seems to have a mechanism for being actively imported into the nucleus. It does not have to wait for a breakdown of the nuclear envelope during cell division, as it is required for other retroviruses. Hence, retroviruses only transduce dividing cells, while the lentivirus transduces dividing and non-dividing cells.

Lentiviruses have a few nuclear-import mechanisms, not all are clearly understood. There is a defined nuclear localization signal in the matrix region of the lentivirus gag protein that is important (MLV retroviruses do not have this), but there are other mechanisms involved as well that are not yet defined. Just putting a nuclear localization signal into MLV gag does not make it transduce non-dividing cells.

Lentivirus vs. Adeno-Associated Virus (AAV): (disadvantages of AAV) (back to Table of Content) (back to Protocol and Application Notes) (back to Virus and vector biology)

It’s not clear that AAV integrates into the genome in all cases. Through gene therapy studies, it has been shown that other nearby chromosomal rearrangements can occur

when AAV integrates, and this has not been shown to be true for other integrating viruses such as Lentivirus.

AAV system has more packaging constraints than the Lentiviral system. Both of these viruses should be used in a BL2 facility but our lentiviral system has additional safety

features that are not in the AAV system: Addition of the REV gene in the packaging mix, so that it is required to transport the viral RNA

out of the packaging cell line and into the cytoplasm so it can be packaged. Hence if no Rev is expressed, no viral transcript is packaged.

Expression of the gag/pol gene required for packaging also requires the presence of REV, so this is more added safety.

A 5' self-inactivating feature which eliminates the 5' viral LTR following integration into the host cell.; There will not be transcription from the viral promoter.

Physical Characteristics (based on HIV) (back to Table of Content) (back to Protocol and Application Notes) (back to Virus and vector biology)

Morphology: Virions have a complex construction and consist of an envelope, a nucleocapsid, a nucleoid, and a matrix protein. Virions are enveloped, spherical to pleomorphic in shape and have a size of 80–100 nm in diameter. The surface projections are small or inconspicuous spikes that are densely dispersed covering evenly the surface. Surface projections are 8 nm long. The core is rod-shaped, or is truncated cone-shaped. The nucleoid is concentric.

Physicochemical and Physical Properties: Virions have a buoyant density in sucrose of 1.13–1.18 g cm–

3. Virions are sensitive to treatment with heat, detergents, and formaldehyde. The infectivity is not affected by irradiation.

Proteins: Proteins constitute about 60% of the particle weight. The viral genome encodes structural proteins and non-structural proteins. Virions consist of 5 major structural and 3 non-structural proteins. The virus codes for an RNA-dependent DNA polymerase.

Lipids: Lipids are present and located in the envelope. Virions are composed of 35% lipids by weight. The composition of viral lipids and host cell membranes are similar. The lipids are of host origin derived from plasma membranes.

Carbohydrates: Three percent of the particle weight is attributed to carbohydrates. 293FT Producer Cell Line

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(back to Table of Content) (back to Protocol and Application Notes)

General Information Culturing 293FT cells Supplementing 293FT culture medium with Sodium Pyruvate

General Information

F stands for fast-growing cells T stands for the larger T antigen. The plasmid expressing T antigen is stably integrated in the 293FT cells. It

has the large T antigen, but no SV40 origin of replication. Thus, it would not survive as an episome. It is recommend not to use this cell line for generating lentiviral stable transfectants due to the presence of T

antigen, which would recognize the SV40 ori present on Invitrogens lentiviral vectors and attempt to induce DNA replication at the site of integration, resulting in cell death. This applies to 293FT cells and pLenti4 & 6 vectors and not to pLenti6.2 vectors. The pLenti6.2 vectors do not have an SV40 promoter driving the bsd-R gene; it is the PGK promoter. 293 FT cells can be transduced with virus made from pLenti 6 or 4 vectors and pLenti 6.2 vectors, but it is strongly suggested that 293 FT cells be used for virus production and HT1080 cells for titer determination. For additional information see Tips to Improve Viral Titers.

HEK 293 cells can be used instead of 293 FT; however, the titer will be lower. The large T-antigen helps to prevent integration of the Lentiviral expression vector into the 293-cell line genome. Thus, all transfected Lentiviral vector DNA remains available for virus production. It is not recommended to T-Rex 293 cells instead of 293FT cells to produce virus. In the absence of tetracycline, the repressor protein TetR, will bind and sterically hinder/repress transcription from the internal CMVTO promoter. This will potentially reduce virus titers due to steric hindrance of transcription from the RSV/5'LTR (which must read through CMVTO to generate the viral genome). This will still result in translation of the (toxic) Gene of interest (GOI). However, translation of the GOI may be significantly reduced enough to allow generating of some virus titers (especially if there is a threshold at which the GOI induces toxicity).

There is no evidence that TetR interferes with Rev function: TetR binds DNA at the Tet operator to inhibit transcription; Rev binds and exports RNA to the cytoplasm for translation and/or encapsidation.

Culturing 293FT cells (back to Table of Content) (back to Protocol and Application Notes) (back to 293FT Producer Cell Line)

Thawing – Things to consider when thawing 293FT cells and cells look unhealthy: Wash the thawed

cells once with PBS before plating (i.e. quickly thaw the cells, and immediately after thawing, transfer the cells to a 15 ml tube containing 10 ml PBS, and then pellet the cells). Resuspend the cells in media and plate in media without Geneticin (G418). Leave the cells in media without Geneticin overnight, and the following day, replace with media containing Geneticin. It is not necessary to grow thawing cells in media with 20 % FBS; we normally use the standard 10% FBS

When thawing 293FT cells allow the cells to attach overnight in regular DMEM media (+10% FBS) in the absence of G418. Twenty-four hrs later replace media with regular DMEM in the presence of G418. 293 cells may come off in sheets if not cultured or handled properly especially after transfection. 90% of the cells should remain attached during the entire viral production (even with lots of syncytia) if they are plated as recommended and they are treated gently. Alternatively, the cells can be plated on "coated" 100mm plates (e.g. poly-lysine, Biocoat, fibronectin, etc.) but these types of plates can be expensive if a lot of virus production is being done. If the cells are kept at room temp too long (or if incubator is not actually at 37 degrees C) they will detach very quickly.

If 293 FT cells lift off shortly after transfection, i.e. 18 h posttransfection or even earlier, it indicates that cells were not 90% confluent and the Lipofectamine 2000 induces toxicity. If cells lift off 24 or 48 h posttransfection then this is not really a bad sign. The cells may have not been treated gently enough or may have been seeded too densely. At this point virus can be harvested according to instructions in the manual.

Handle the dishes of cells gently, no swirling or sloshing around while moving the plates.

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Squirting the cells off instead of trypsinization is NOT recommended. Pipetting off cells selects for cells that detach easily and when these cells are re-plated, they are attached to each other more tightly than to the plastic dish and they tend to come off in sheets.

Trypsination: The Trypsin used is 0.25% (1X) cat# 15050-065, 2ml/100mm plate and incubated for 2 to 3 minutes at room temperature. The Trypsin reaction is stopped with 10 ml of media (OptiMem with 10% FBS or regular growth medium without any antiobiotics) and then centrifuged for 5 min at 1500rpm. If plating on the day of transfection you need to have at least 5 million cells/ 100mm plate. If plating on the day before transfection you need 5 million /plate. Sodium pyruvate is not required for culturing these cells but there is a reproducible slight but significant improvement in lentivirus production from 293FT cells grown in the presence of sodium pyruvate. In some cases it could be the difference between unacceptably low versus satisfactory viral titers, see also details in the next chapter: Sodium Pyruvate. 293FT cells are not tested for blood-borne pathogens. They are only screened for bacteria and mycoplasma.

Any syncytia formation during regular culturing of the cells could be a stress response to lack of the appropriate nutrients in the media. In these situations check either (1) CO2 levels; (2) media composition and stability (including making sure non-essential amino acids and sodium pyruvate is added to the media).

Check that the CO2 level in the incubator is 10%. Prevent acidic culture conditions by increasing the buffering capacity of the media by adding Hepes buffer to 25-50 mM final concentration after transfection.

The high acidity in the media could affect cell health and viral production; and too high an acidity reduces viral infectivity. Note: even if one assays for viral structural proteins in acidic media using the p24 assay one can see high level production of viral particles; however most of these would not be infectious since too high an acidity can reduce viral infectivity

When the cells are healthy, even under acidic media, 10ml media is replaced after 24h with fresh 10ml media, then this second batch of media 24h later will have a lower viral titre compared to the first batch of medium. If the first 10ml goes for 48h then the viral titre will be equivalent to, or only slightly less than, the amount collected in 20mls (one 10ml at 24h and another 10ml 24h later). .

Supplementing 293FT culture medium with Sodium Pyruvate (back to Table of Content) (back to Protocol and Application Notes) (back to 293FT Producer Cell Line)

Pyruvate is an intermediary organic acid metabolite in glycolysis and the first of the Embden Myerhoff pathway that can pass readily into or out of the cell. Addition to tissue culture medium provides both an energy source and a carbon skeleton for anabolic processes. Addition to 293FT cultures (1mM) generally ensures a healthier culture and consequently improved titers.

Addition may help in maintaining certain specialized cells, may help when cloning and may be necessary when the serum concentration is reduced in the medium (Culture of Animal Cells: A Manual of Basic Technique, 3rd edition, (1994) by R. Ian Freshney (Wiley- Liss, Inc., New York).

May also help reduced fluorescent light-induced phototoxicity (see below) Usually sodium pyruvate is added to give a final concentration of 0.1 mM. Sodium pyruvate is available as

a 10 mM (100X) stock solution. GIBCO #11360070 An important but often overlooked source of chemical contamination results from the exposure of media

containing riboflavin or tryptophan to normal fluorescent lighting (see references below). These media components are photoactivated by UV radiation emitted from most fluorescent lights and give rise to hydrogen peroxide. This generates free radicals that are toxic to cells; the longer the exposure the greater the toxicity. HEPES (an organic buffer commonly used to supplement bicarbonate based buffers) appears to increase the phototoxic exposure effects (2, 3).

Sodium pyruvate has been shown to reduce or eliminate this phototoxicity (3). Short-term exposure of media to room or hood lighting when feeding cultures is usually not a significant problem. However, leaving media on lab benches for extended periods, storing media in walk-in cold rooms with the lights on, or using refrigerators with glass doors where fluorescent light exposure is more extensive will lead to a gradual deterioration in the quality of the media.

Wang, R.J. (1976) Effect of Room Fluorescent Light on the Deterioration of Tissue Culture Medium. In Vitro 12:19.

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Zigler, J.S., et al. (1985) Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro 21: 282.

Spierenberg, G.T., et al. (1984) Phototoxicity of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid-buffered culture media for human leukemic cell lines. Cancer Research 44: 2253.

Experimental Procedures (back to Table of Content) (back to Protocol and Application Notes)

Overview Cloning Transformation

Transformation with Stbl3 E.coli Transformation with One Shot ccdB Survival T1 Phage-Resistant Cells

Transfection Pre-testing of Concentration expression vector before transfection for virus production Transfection of 293FT cells Virus production and harvest of Virus Concentration of Virus Titering viral supernatant Polybrene Tips to Improve Viral Titers Packaging Limits

Transduction Difference between Lentiviral and Adenoviral transduction FACS analysis with GFP-lenti transduced cells Transduction of virus in vivo and in tissue slices Stable integration of lentiviral constructs Creation of a Double Stable Cell Line Transient transduction Leakiness of expression B Lymphocytes T lymphocytes Transduction of Suspension Cells Monocytes and Macrophages Transduction of Neurons Embryo transduction Transduction in Chickens Optimizing Lentiviral Transductions

Troubleshooting for Protein Expression Virus rescue

Overview (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Clone gene into Lentiviral vector. Transform E coli and isolate plasmid Test construct by transient transfection of plasmid and expression from construct Transfect 293FT cells with construct and packaging mix. Harvest and titer viral stock using HT1080 cells. Transduce target cells with virus.

Cloning (back to Table of Content)

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(back to Protocol and Application Notes) (back to Experimental Procedures)

When cloning into pLenti/D-TOPO vectors, a proofreading polymerase needs to be used for generation of the PCR product. The mix of Taq polymerase and proofreading enzyme is NOT recommended since the presence of 3’-A overhangs will inhibit the D-TOPO reaction.

Ensure that 1:1 ratio of insert:vector is maintained. An excess of PCR product can inhibit the D-TOPO reaction.

Generally background colonies with pLenti-D-TOPO vector alone should be low. All retroviral vectors contain two LTRs that are around 180 basepairs of perfect repeats; hence they can

easily recombine in bacteria resulting in a smaller product. Invitrogen vectors have a low percentage of recombinants. During the LR reaction: Between pLenti-DEST vectors and the Entry clone DNA there is a possibility of an inappropriate recombination event occurring that may result in a shorter sized, less than 6kb plasmid. When pLenti6/DEST DNA was tested in an LR reaction, it was shown to have less than 5% LTR recombinants. If greater than 5% of LTR recombinants is observed, then either there was excess of the Entry clone DNA or insufficient LR clonase in the LR reaction. In either scenario there is a high possibility of co-transformation with both the Entry and the DEST plasmids in a single colony.

Transformation (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Stbl3 cells are strongly recommended for cloning to reduce the likelihood of in appropriate recombination between the LTRs.

Even strains that greatly reduce recombination cannot prevent it completely. It is strongly recommended to analyze transformants with restriction enzymes that cut within the LTRs and/or the RSV promoter region, to make sure that the elements required for virus production are intact, as well as enzymes to confirm cloning of the gene of interest. Another validation of the pLenti expression vectors is to digest them with BsrGI (recognition site resides in the att sites) and with Afl II (recognition site resides in the LTR sites). Any unwanted recombination in the LTR sites will disqualify the pLenti vector, see also Troubleshooting for Protein Expression and Tips to Improve Viral Titers.

Original suggestions were to plate on ampicillin and blasticidin to avoid growth of recombinants. However, since dosing of two antibiotics proved to be problematic in some situations, recent protocols favor the use of a single antibiotic, ampicillin, and picking only small colonies. The recombination, which results in loss of most of the plasmid, confers a growth advantage on the cells and the colonies are very large. The smaller colonies contain the entire plasmid. Selection can also be done on plates containing Bsd and Amp as described in the manual. It is very important to not use low salt LB plates and to not grow liquid cultures in medium containing Bsd. If low salt LB medium is used no colonies will be observed since Bsd is more active in this medium.

In case Bsd is spreaded out, R&D recommends to spread out as final concentration 15 ug per ml LB agar ( = 15 ug Bsd/ml). Thus, if the plate contains 30 ml LB agar, one would spread out 15 ug x 30 = 450 ug. If the Bsd stock is 1 mg/ml and the LB plate contains ~ 30 ml LB agar, one would spread out 448 ul or just 450 ul Bsd stock onto a-plate containing ~ 30 ml solidified LB agar. According to R&D, one would let the plate incubate for 30 min at 37oC prior to use in order to ensure good penetration of the antibiotic into the agar. One should remember that the selection with 100 ug/ml ampicillin with Stbl3 is the general or first recommendation.

Transformation with Stbl3 E.coli (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

This strain is endA+. A very thermostable periplasmic endonuclease will co-purify with the plasmid DNA, possibly shearing the plasmid during restriction enzyme digestion since the endonuclease needs Mg for activity. The procedure should be terminated with a phenol/chloroform extraction or purifying the

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plasmid DNA on a column. If making solutions I, II, and III (alkaline Lysis, see Maniatis or Current Protocols for Molecular Biology) for a conventional DNA prep procedure, make sure EDTA is present in solution I. Invitrogens PureLink HQ Miniprep kit (K2100-01) does have EDTA in solution I (10 mM EDTA) and should work well with Stbl3 cells, see parapgraph below when to make a pLenti vector mini or midi prep.

Small colonies may be observed with Stbl3 E. coli but typically the colonies are not tiny; they are good-size colonies. Do not plate too many cells per plate

Different-sized colonies and some mini-preps that show LTR recombination may be observed when using the Stbl3 cells. If this occurs pick several colonies of different sizes (i.e. at least 5 small colonies, 5 medium-size colonies, and a few large ones) and analyze them with restriction enzyme digestion.

Another option is to do double antibiotic selection on the LB plates with both Amp and Bsd. Remember: first recommendation is selection with 100 ug/ml Ampicillin.

Transformation with One Shot ccdB Survival T1 Phage-Resistant Cells (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

These cells are not recommended for propagation of vectors with direct repeats. Lentiviral vectors give very low yields with these cells, which may become problematic. It is recommended to use DB3.1 to propagate lentiviral gateway vectors. If ccdB-survival cells must be used, the following is recommended to reduce the chance of recombination between direct repeats (LTR):

Select and culture transformants at 25ºC-30ºC. Do not use “richer” bacterial medias such as TB since these media tend to give rise to a greater

number of unwanted recombinants, see above. If the plasmid confers chloramphenicol resistance, select and culture transformants using LB medium containing 15-30 µg/ml chloramphenicol and an additional antibiotic appropriate for selection of the plasmid.

Transfection Pre-testing of expression vector before transfection for virus production (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures) The lentiviral expression vector can be pre-tested by transfecting the vector into the cells of your choice. This is a quick way to test if the GOI is toxic. In this case the polyA signal in the 3’LTR is used. Transfection of 293FT cells (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

It is strongly recommended that no G418 be added during transfections or at any time during virus production since this can result in a lot of cell death.

It is believed that it is possible to transfect 293FT cells in suspension to package virus. However currently there is no validated protocol for packing virus in suspension.

239FT will easily adapt to most (if not all) serum-free formulations. However 293 SFM II is probably not the best choice because of poor transfection efficiency in this media. The FreeStyle 293 Expression medium is recommended. Advanced DMEM or OptiMEM with reduced serum are also both good choices. OptiMEM with serum has been routinely used for transfection to produce virus.

Lipid:DNA complexes can be added to cells or lipid:DNA complexes and 293FT cells simultaneously can be added to the plate. Also see Tips to Improve Viral Titers.

Expression of the VSV-G glycoprotein causes 293FT cells to fuse, resulting in the appearance of multinucleated syncytia. This morphological change is normal and does not affect production of the

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lentivirus. If syncytia are not observed, virus production is likely to be impaired. However one can process with the virus production as outlined in Tips to Improve Viral Titers.

The next day after the transfection, typically some rounding up of the cells will be observed but the cells usually recover after removal of the transfection medium and addition of fresh medium.

Toxicity is significantly reduced if cells are plated at 90% confluence. If they are less than 90% confluent then there probably will be a lot of toxicity and low virus production (5-6 x 106 cells per 10 cm2 should give roughly 90% confluence on the day of transfection).

In our experience, the presence of serum during transfection will improve virus production since the cells will be healthy and transfect really well. .

Adherent cells in T175 flasks can be used for scaling up the virus instead of using suspension cells: Seed 1 x 107 cells/flask overnight; transfect with 6 ug Lenti + 18 ug Packaging Mix using 72 ul Lipofectamine 2000 lipid. Treat and harvest following the regular Lentiviral Protocol.

To scale up to a T225 flask find the relative growth area of the T225 relative to a 100 mm plate (say Y-folds).

Multiply 5 x 106 (the number of cells to plate in the 100 mm plate) by Y to get the number of cells to plate in the T225 plate.

For amounts of DNA and LF2K to use in T225, multiply 3 by Y (for microgram pLenti vector to use) 9 by Y (for microgram Packaging Mix) 36 by Y (for ul of Lipofectamine 2000 to use). These proportionalities apply to any plate size.

Virus production and harvest (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Harvest virus-containing supernatants 48-72 hours post-transfection. 293FT cells producing virus generally form syncitia. Usually, the cells do not detach from the plate, but it

can happen 24-48 h posttransfection. If this happens, proceed and harvest the virus as recommended in the manual. The cells come off easily if not treated gently.

Caution: Remember that you are working with infectious virus at this stage. Follow the recommended guidelines for working with BL-2 organisms (see the manual for more information).

Centrifuge at 3000 rpm for 15 minutes at 4°C to pellet cell debris, filter if desired. Store supernatant in aliquots at -80°C.

Viral titer is not significantly different if post-transfection time is counted from the point of adding DNA/lipid or from the point of removal of DNA/lipid mixes.

The presence of IRES between two genes cloned into the pLenti vector should not interfere with titer or packaging as long as the insert is within packaging size limits (~5.5 KB). Typically genes downstream of IRES tend to be expressed at a lower level than the upstream gene. Hence the time of expression may have to be extended.

It is strongly recommended that the Lentivirus be aliquoted immediately after production and stored at –80oC. This way, every time a new aliquot is thawed it will be the same titer as the first tube (Keeping the Lentivirus overnight at +4oC is not ideal, but still alright). Lentivirus is more sensitive to freeze/thaw than Adenovirus. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while Lentivirus can lose up to 5% or more of its titer with each freeze/thaw. When stored properly viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, it is recommended the viral stocks be re-titered before use.

Concentration of Virus (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

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Before doing ultracentrifugation to concentrate virus, it is important to spin down the viral sup (to pellet cells), and then filter the viral sup through a 0.45 micron filter to remove any contaminating cells. Lentivirus will pass through a 0.45-micron filter but will be retained on a 0.22-micron filter.

Recommended literature about Lentivirus concentration via ultracentrifugation :- Reiser J, (2000). Production and concentration of pseudotyped HIV-1-based gene transfer vectors. Gene Ther. 7:910-913.

Burns et al, (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037. The non-concentrated virus can be kept in complete medium that is used to culture 293 FT cells during

virus production. This complete medium is composed of D-MEM with 10% FBS, 2 mM L-glutamine, 0.1 mM MEM Non-essential Amino Acids, 1% pen/strep, and 1 mM sodium pyruvate

Non-concentrated and concentrated virus is best kept in small aliquots at –80oC. Titering viral supernatant (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures) Also see Tips to Improve Viral Titers.

Titering is most often done by determining the number of antibiotic resistant cells (colonies) that arise after transduction and selection.

For HT1080 cells, the typically used dose of blasticidin is 10 ug/ml, but in general one should generate a kill-curve for each antibiotic and cell line before proceeding. Most cell types respond between 1 ug/ml and 10 ug/ml.

HT1080 human fibrosarcoma cell-line (ATCC #CCL-121) is recommended for titering lentivirus. The titer can be 10-fold less if using HeLa or NIH3T3 cells.

The transient expression method of titering is not recommended, as it is neither sensitive nor accurate. During titering, the number of virus that can transduce an antibiotic selection gene into HT1080 cells is

being determined. Since the antibiotic resistance gene is in cis with the gene-of-interest, the titer gives the number of viruses that will also transduce the gene of interest. Crystal violetstains proteins in the cells that survive the antibiotic selection; the mock transduction shows no staining because the cells have died and detached from the plate during selection.

Human cell lines tend to give higher viral titer. The CMV promoter might be shut off in BHK and other rodent cell-lines.

Using 293FT for titering is not recommend, since most the Lentiviral vectors contain the SV40 early promoter and ori. The exceptions to this are the pLenti 6.2 vector because they contain the PGK promoter instead.

Titering of HT1080 cells kept either under Zeocin or Blasticidin (bsd) selection. Fortyeight hours post-transduction, the cells were placed under 10 ug/ml blasticidin or 100 ug/ml zeocin selection, as appropriate. In particular, Zeocin selection was done as follows: 24-hour post-transduction cells were trypsinized from 6-well plates and expanded into 100mm plates. 24 hrs after expansion into 100mm plates, 100 µg/ml Zeocin was added to the transduced cell culture medium for selection. After 7 to 10 days of blasticidin selection, or two to three weeks of zeocin selection, the resulting colonies were stained with crystal violet : A 1% crystal violet solution was prepared in 10% ethanol. Each well was washed with 2 ml PBS followed by 1 ml of crystal violet solution for 10 minutes at room temperature. Excess stain was removed by two 2 ml PBS washes and colonies visible to the naked eye were counted to determine the viral titer of the original supernatants.

Titering based on GFP expression – For use FACS to determine titers based on GFP expression, the following reference should be helpful as it has a procedure and formula for calculating titers based on GFP expression. The reference is: Sastry L et al. 2002 in Gene Therapy (2002) 9, p. 1155-1162 Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

Polybrene (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

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It is a small, positively charged molecule that binds to cell surfaces and neutralizes surface charge and greatly enhances transduction by retroviruses.

Apparently allows the viral glycoproteins to bind more efficiently to their receptors, because it reduces the repulsion between sialic acid-containing molecules.

The cells that are to be infected should be pre-treated with polybrene and virus adsorption should be done in the presence of polybrene.

Polybrene can be added to the viral dilution before adding the virus to the cells. However the amount added must dilute out to 6ug/ul in the culture medium after transduction.

Cells vary in the amount of polybrene that they will tolerate. Typically it is between 1 to 10 µg per ml. Depending on the cells being transduced, polybrene may or may not affect transduction efficiency;

however in most cells it can increase transduction efficiency anywhere from two-to-ten fold. Polybrene can be toxic to terminally differentiated neurons and dendritic cells. In situations like this,

titration of polybrene using 1,2,4,6 ug/ml will be have to be done to determine the highest nontoxic concentration that can be used. If toxicity is a big problem, then cells can be transduced in the absence of polybrene but the MOI will have to be increased by twofold.

Another alternative if Polybrene is toxic during transduction is the use of DEAE-dextran , see below. DEAE-dextran

DEAE can be used instead of polybrene during transduction, see below. Diethylaminoethyl-Dextran (DEAE-Dextran) is a polycatonic derivative of Dextran

Reference: Reiser et al. PNAS, Vol 93, pp. 15271, 1996. In this paper cells were transduced with DEAE

or polybrene. Tips to Improve Viral Titers (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures) Good lentiviral titers begin with optimal transfection efficiencies. Below are some suggestions to help optimize transfection efficiencies when working with Invitrogen’s lentiviral products.

I. 293FT Cells Use low passage 293FT cells. Do not use 293FT cells over passage 20. We suggest growing 293FT for

enough passages to freeze down many aliquots, then taking out as needed. Grow for 2-4 passages, then use for viral packaging.

Passage cells in complete DMEM containing G418 (500 ug/ml). Supplement the media with "non-essential" amino acids and sodium pyruvate, (0.1 mM MEM Non-Essential amino acids and 1 mM MEM Sodium Pyruvate). Culture always with 10% FBS cat# 16000-044.

Plate cells at 5 x 106 per 100 mm dish - cell density is very important. Make sure the cells are growing well before re-plating prior to the day of transfection - avoid overgrowth of 293FT cells when passaging.

When plating for transfection the next day, do not add any antibiotics (i.e. G418/Geneticin or pen-strep) to the media

Use 10% FBS cat# 16000-044 for 293FT cell culture during 293FT transfections for virus production, and during lentivirus transductions. Our in-house studies have indicated that other types of serum may adversely affect 293FT transfection efficiency and/or lentivirus particles, resulting in decreased viral titers

II. Lipofectamine 2000 & Plasmid DNA

Lipofectamine 2000 reagent – make sure the reagent has been stored properly and do not vortex the reagent tube – mix gently by inversion. Additionally, treat lipid:DNA complexes gently, do not pipet up and down.

Quality of plasmid DNA - plasmid DNA isolated with Invitrogen’s S.N.A.P. columns (midiprep) or PureLink maxi or mega kits is sufficiently clean for these transfections. Do not use plasmid DNA from minipreps.

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III. Transfection tips 293 FT cells should grow in medium containing 10% FBS (cat# 16000-044); . Seed 5 million cells the day

before transfection, incubate overnight following instructions in the manual. Make sure cells are about 90% confluent before transfecting. (Note that the same-day cell plating and transfection method described in the manual may give slightly higher titers, but it is also designed for experienced users, as it can be more difficult).

In general, 293 FT cells do not detach during lentivirus production - The majority of 293 FT cells usually do not detach between 24-72 h post-transfection (although this may happen in some cases, as mentioned below). The 293 FT cells typically become multinucleated (syncitia) and look plump due to VSV-G expression. If 293 FT cells do not show any signs of syncitia and do not look plump, virus production is likely to be impaired; nevertheless proceed with the virus production. If titer is low, concentrate the virus.

If the majority of cells are detached between 24-72 h post-transfection, it is not necessarily a bad sign, since expression of some genes from the lentiviral vector may make the cells detach more readily; in this case, harvest the virus and determine the titer. Detachment of cells could also be due to cells not being treated gently enough (they come off very easily) or that they have been seeded far too densely, i.e. far beyond 5- 6xE6 cells/10 cm plate.

On the other hand, if the majority of cells detach during or shortly after transfection, i.e. less than 18 h post-transfection, it is a sign of toxicity. Most likely it means the Lipofectamine 2000 is toxic because the 293 FT were less than 90% confluent on the day of transfection. A low titer is then expected.

Invitrogen offers the pLenti6.2-GW/EmGFP vector (V369-20) that serves as a transfection control (to measure transfection efficiencies in 293 FT cells) and as a transduction control to determine the appropriate MOI for a given target cell type.

IV. Suggestions for setting up a transfection experiment (set up 4 separate plates):

Plate 1: Lentivirus production Plate 5x 106 293 FT cells; next day add 3 ug Lentiviral vector.+ 9 ug packaging mix + 36 ul Lipofectamine 2000 as recommended in the manual. Leave the transfection medium on the plate overnight and replace next morning with regular growth medium lacking antibiotics.

Plate 2: FACS Analysis (to assess transfection efficiency) or Western (to confirm expression)

Plate 5x 106 293 FT cells; next day, add 3 ug lentiviral vector + 9 ug irrelevant “filler” DNA (i.e. empty expression vector like pcDNA in order to ensure highest biosafety) in place of packaging mix (without packaging mix, virus will not be made) + 36 ul Lipofectamine 2000. (For instance, pLenti6.2-EmGFP or any other GFP Lentiviral expression vector could be used). Leave the transfection medium on the plate overnight and replace next morning with regular growth medium lacking antibiotics.

Plate 3: Lentivirus production

Set up the same conditions as Plate 1, except replace media with regular growth medium lacking antibiotics at 4-6 hrs post-transfection.

Plate 4: Negative control for Lentivirus production

Plate 5x 106 cells 293 FT cells. Do not add any DNA/Lipid complexes (could do mock transfection either using lipid or packaging mix only). Leave the transfection medium on the plate overnight and replace next morning with regular growth medium lacking antibiotics.

One may also try slightly increasing the DNA and LF2K amounts proportionately as follows for a 100 mm plate (maintain the same ratio of Lipofectamine 2000:total DNA at 3:1), and remove the transfection complexes after 4-6 hrs. Try the following amounts: 4.5 ug expression vector & 18 ug packaging mix + 67.5 ul Lipofectamine 2000 Alternative: 5 ug lenti vector + 15 ug packaging mix + 60 ul Lipofectamine 2000 Harvest the first and second plate 48 h or 72 h posttransfection. Harvest the third plate 72 h posttransfection. Do not change the medium after 48 h post-transfection if harvest is 72 h post-transfection. Add a little bit of medium if the medium turns too yellow or if 293 FT cells seem to starve in plate 4 (negative control).

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V. Determination of Viral Titer:

If proceeding to transduction it is strongly recommended to determine the virus titer in HT1080 cells. If the lenti vector contains GFP, FACS analysis can be performed for GFP expression to determine the titer. Reference for GFP titering method: Gene Therapy (2002) 9: 1155-1162. If the lenti vector does not contain GFP, use the antibiotic selection titering protocol in the manual (blasticidin for pLenti 6 vectors or zeocin for pLenti 4 vectors).

With lacZ lentivirus one can get an approximate idea of the titer (based on lacZ expression following transductions at different viral dilutions), but this is not a very quantitative titering method. Based on our experience we have found that LacZ titering is not nearly as accurate or reproducible as titering based on GFP expression or Bsd selection, since one has to rely on counting blue cells by eye under the microscope.

If using LacZ staining reagents, it is possible that the conditions (i.e. X-gal concentration) were previously optimized for detection of transiently transfected plasmids, which will be present at very high copy numbers and thus easy to detect. With lacZ lentivirus transductions for titering, on the other hand, there is a possibility of only getting 1 virus particle per transduced cell. Therefore it may be worthwhile to increase the X-gal concentration and staining time, and maybe also try using more virus if possible. Also allow more time for virus integration into the genome; wait until 48-96 h post-transduction to assay for LacZ activity (Cells should be incubated with the virus for 24 h, the virus should be replaced with regular growth medium, and then 24 to 72 h later, cells can be fixed for LacZ staining).

It is strongly recommended that HT1080 cells be used to determine lentivirus titers. This helps with standardization of titers obtained between different users. The transduction efficiency is high in these cells, and titering results are very accurate and reproducible, making HT1080 cells the gold standard for accurate titering. Different MOIs in other cell types can be used based on HT1080 titers. The HT1080 cell line is a human fibrosarcoma cell line and is available from www.atcc.org (ATCC Catalog number:.CCL-121).

The titer of 4.8x 106 cfu/ml for the lacZ virus mentioned in the Lentiviral Expression manual refers to concentrated virus.

Recombination of the propagated pLenti vector/insert can lead to low viral titers. Digestion with Afl II can be used for clone validation; the recognition sites reside in the 5’ and 3’ LTRs. Afl II is a good diagnostic digest, but note that in some cases (depending on the gene of interest (GOI) size) it needs to be used in combination with another enzyme such as Xho I in order to get bands with sizes that can be distinguished from each other. Xho I is commonly used in-house in a double digest with Afl II since Xho I cuts downstream of the gene of interest, it is a good cutter that uses the same buffer as Afl II, and is unique in most pLenti vectors as long as it doesn’t cut in the GOI. Also, Xho I when used together with Afl II, typically gives band sizes that can easily be distinguished. Another helpful restriction enzyme is BsrGI that cuts into att sites that are part of Gateway vectors. Note that it’s important to check whether the enzymes cut in the GOI.

Packaging Limits (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Lentiviral titer decreases with increasing insert size. The packaging limits for lentivirus are around 6 to 6.5 kb and for adenovirus around 7 to 7.5 kb. Above these limits no virus gets made.

An insert of 5.2 kb with good titer (~0.5 x 10e5 cfu/ml) has been effectively packaged. However, when attempted to package inserts of 6.2 kb or larger, the titers dropped precipitously and the viruses that were generated carried viral genomes that had been truncated and were no longer carrying the GOI (they were only carrying the Bsd gene).

Transduction Difference between Lentiviral and Adenoviral transduction (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

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Adenovirus requires the Coxsackie-Adenovirus Receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the Multiplicity of Infection (MOI) used easily modulates the efficiency of transduction for both viral systems. One also should keep in mind that Adeno vs Lenti transduction efficiency also depends on the target cell.

For cells that have sufficient expression of the CAR receptors and are actively dividing, it should be possible to get adenovirus transduction efficiencies in the range of 80-90%, as long as an adequate MOI is used (for instance, in HT1080 cells, which are readily transducible with adenovirus, transduction efficiencies are around 90% with an MOI of 1). Similar transduction efficiencies are possible with lentivirus in certain cell types (for instance, in HT1080 cells, which are readily transducible with lentivirus as well as adenovirus, an MOI of 1 gives transduction efficiencies of around 90%). There is definitely variability in the transduction efficiencies of different cell types, for both adenovirus and lentivirus. For instance, in some cell types, a 10-fold higher MOI may be needed to get the same transduction efficiency.

In the presence or absence of a selection marker, the adenovirus does not integrate into the genome. The lentivirus, however, does integrate into the genome. It can integrate in the absence of any selection marker and even stronger in the presence of a selection marker, see below under “Transient Transduction”. The adenovirus has a terminal protein (TP) that is covalently linked to the ends of the Adenoviral DNA thereby enabling the adenoviral genome to be very stable in the nucleus (For more details, see in “ViraPower Adenoviral Expression System” Technical Support Note).

FACS analysis with GFP lenti transduced cells (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

A good citation for doing FACS analysis with GFP-Lenti-transduced cells is the following reference- Sastry L et al. 2002 in Gene Therapy (2002) 9, p. 1155-1162 Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Methods. This paper has a procedure and formula for calculating titers based on GFP expression.

Transduction of virus in vivo and in tissue slices (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

This should be feasible since there are many reports in the literature of researchers using lentivirus to transduce mouse or rat organs in vivo. No guidelines regarding this application are available but it is recommended to start with conditions similar to those used in vitro. The virus will most likely need to be concentrated and optimization will likely be necessary.

Dull, et. al. (1998) J. Virology, 72:8463-8471 Naldini et al (1996) PNAS, 93:11382-11388 Miyoshi, et. al. (1997) Proc. Natl. Acad. Sci. USA, 94:10319-10323. Kafri, et. al. (1997) Nat. Genet., 17:314-317 Gouze et al (2002) Mol Ther. 5: 397-404 Amado and Chen (1999) Science, 285: 674-676 Man et al (2005) Ann Plat Surg. 55: 81-86

Stable integration of lentiviral constructs (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Select for stably transduced cells using blasticidin. This requires a minimum of 10-12 days after transduction, but allows generation of clonal cell lines that stably express the gene of interest. For

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NIH3T3 cells use 5 ug/ml of blasticidin for selection. For antibiotic recommendation on other cell lines, see cell lines database on the Invitrogen website.

Stably transduced cells are maintained under blasticidin selection at all times. Creation of a Double Stable Cell Line (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Co-transduction can be performed by selecting for double-resistant populations. It is recommended, but not necessary that transductions are being performed sequentially. First, one selects for one highly expressing antibiotic-resistant clone (e.g. blasticidin). Then, one follows with the second transduction (e.g. zeocin) and selects for those bsd/zeo resistant clones that are now high expressors for gene 2.

We have successfully generated double-stable cell lines (in 293 cells), using sequential transductions with 2 different lentiviruses expressing 2 different genes and antibiotic selection markers. We have tried several different combinations of antibiotic resistance genes, including Hygromycin and Zeocin, or Blasticidin and G418 (Geneticin), and have had good success. It may help to decrease the concentrations of the two antibiotics used for selection. It is a good practice to maintain the selection with the first drug while starting to add the second drug, and it may help to decrease the concentrations of both drugs or at least the first drug, by about 2-fold. This is probably because some of the drugs may have more toxic effects when used in combination than when either one is used by itself.

Transient transduction (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Transient expression can be assayed after transduction of the Lentivirus into mammalian cells Lentiviral transient transduction is typically defined as lentiviral expression without antibiotic selection, which

is quite different from transient transfection of plasmids. During transient transduction, expression of the gene can persist over time even without antibiotic selection, although there will be some variability in expression from cell to cell depending on the integration site. Some low level expression may be observed prior to lentiviral genome integration but the majority of expression will occur after integration. In contrast to plasmids that randomly enter the nucleus, lentivirus targets the nucleus where it only integrates the portion, typically containing the gene of interest, between the 5' and 3' LTRs into the genome. Transfected plasmids tend to integrate less efficiently, and generally only in the presence of antibiotic selection. There is a possibility of integration occurring in the middle of the gene of interest or promoter region, in which case no expression will be observed. In addition, mammalian cells tend to lose the integrated construct in the absence of antibiotic selection.

It is recommended to wait a minimum of 24-48 hours after lentiviral transduction before harvesting the cells to allow the lentivirus genome to reverse transcribe and integrate into the chromosomal DNA.

Leakiness of expression (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures) There is no inherent "leakiness" of expression when the CMV promoter is removed. The 5' LTR is completely inactivated after reverse transcription. However, the site of chromosomal integration in the target cell could provide strong promoter activity that reads through the polyA signal in the 5'LTR, resulting in expression of your gene in the promoterless context. B Lymphocytes (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

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Lentivirus transduction in B-lymphocytes has not been tested in house, Lymphoid cells are notoriously difficult to get anything into but it appears that the issue with B-cells is the

promoter of choice, not the virus. The CMV promoter does not work well in B-cells. There have been reports that with the right promoter (e.g. B29 which is B-cell specific) lentivirus work

fine in B-cells. However B29 with Invitrogen’s Lentiviral expression system has not been tested. B29 is a B-cell-specific member of the immunoglobulin gene superfamily and is expressed throughout B-cell development.

There is a possibility that using the minimal B29 promoter can lead to silencing (Thompson et al; The promoter and 5' flanking sequences controlling human B29 gene expression. Blood 87: 666-673, 1996).

It is believed that there is a possibility that the Ubc promoter will most likely give long-term expression in B-cells. However the expression will be lower than the CMV promoter. One can try to overcome the lowered expression with an increased MOI.

The Ubc and EF1 promoters are good choices for lentiviral transduction of B and T cells, but we have not tested them in-house. So far only human 293 cells and rat neural stem cells have been tested in-house with lentiviral constructs containing an EF promoter. However, we recommend doing literature searches to see if there's any data on how well EF1a or Ubc promoters work in B and T cells.

T lymphocytes (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Transduction of suspension cells such as Jurkat can be relatively inefficient compared to adherent cells such as HeLa and CHO. In this case centrifugation-mediated transduction can be used (Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels. Kuhlcke et al, 2002 Mol Ther. 5:473-478).

If necessary cells can be stimulated; although if transduction is done at high MOI (>5) and the centrifugation method is used, it may not be necessary to stimulate the cells.

In general, the CMV promoter is generally stronger than UbC. The level of expression increases with MOI. However the UbC promoter is not subject to methylation-dependent downregulation.

References

Bai et al. Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector. Gene Ther 2003. 10:1446-1457

Cavalieri et al. Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact Immune competence. Blood.2003, 102:497-505.

Zhou et al. Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells. Hum Gene Ther 2003. 14: 1089.

Sutherland and Williams. Viral promoter expression in CEM-C7 and Jurkat human T-lymphoid cell lines. J Immunol Methods 1997. 207:179-183.

Barry et al. Lentiviral and murine retroviral transduction of T cells for expression of human CD40 ligand. Hum. Gene. Ther 2000, 11:223-232.

Zarrin et al. Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines. Biochem. Biophys. Acta 1999,1446:135-139.

Transduction of Suspension Cells (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Serum free media may be used when performing transduction It has been demonstrated in the literature that greater success is obtained when transducing suspension

cells with either retrovirus or lentivirus in a small volume and gently centrifuging the cells through the supernatant. Somehow this greatly increases the likelihood of the virus contacting the cell and thus increasing the transduction efficiencies.

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Transduction by centrifugation can performed in a 6-well plate or in small volumes. For a 6-well plate with unconcentrated virus:

Plate cells at 1 x 106/ml in a well of a 6-well plate (use 2 ml/well) Add viral supernatant (The cells can be brought up to density in the viral supernatant if the titer is low) Spin at 1000 x g for 1 hr at room temperature (32oC). Return the transduction mixture to 37oC/CO2 incubator for 12 hrs/overnight. Proceed with necessary culture conditions/manipulations The same procedure can be adapted in a 24-well especially if the titer is high (> 1 x 107 cfu/ml) where an

MOI > 1 can be used Reference: Molecular Therapy vol. 5, No. 4, April 2002, Proc. Natl. Acad. Sci. USA vol. 92 pp. 7739-

7743, August 1995 Monocytes and Macrophages (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Macrophages and monocytes although derived from hematopoietic precursors and grow as suspension culture, are not T cells. However the MOIs used to transduce macrophages may be similar to MOIs required for B or T cell transductionsFor suggested MOI ranges, see the table in Optimizing Lentiviral Transductions

More details about the transduction of the difficult to transfect monocytes can be found in: Lennartz, MR, Brannock, PM, and Mazurkeiwicz, JE. Visualization of signal tranduction pathways in real time: protein kinase C and phagocytosis. In: Molecular Morphology of Human Tissues, Techniques and Applications (GW Hacker and RR Tubbs, ed), CRC press, Chapter 15, 2004. In this chapter, the authors describe how the ViraPower Lentiviral Expression system was used to transduce a human monocytic cell line, Mono Mac-6 that is typically a very difficult cell type to transfect. There is also a detailed lentiviral transduction protocol and the authors claim that they did not see expression of their transduced GOI until 7 days post-transduction.

Suggestions for transduction of Human PBMC's in serum free condition: Although this has not been tested in-house, here are some suggestions : The lentivirus can be made in serum, purified and then added back to cells in serum free conditions. The virus can be purified by ultracentrifugation for long-term storage (see: Reiser, J. (2000) Gene Therapy 7:910-913 or Concentration of Virus). After purification the virus can be resuspended in serum-free media that PBMCs are grown in. Add protein to the virus for long-term storage.

Another suggestion is to do the initial 4-plasmid transfection in serum, and switch to serum free conditions for production. Remove the transfection compleaxes the next day and wash with PBS. Feed the cells with a serum-free formulation. Virus can be made under serum-free conditions but the titers will be lower. It is not necessary to add back serum or BSA to the supernatants for long-term storage.

Transduction of Neurons (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures) Lentiviruses certainly do infect non-dividing cells in nature. How they do this is still being worked out in the literature. Primary post-mitotic E18 rat hippocampal and cortical neuronal cells are typically transduced with the concentrated lentivirus. Brain tissues were dissociated, spun down, and resuspended in NeuroBasal Medium with B27, 0.5mM L-glutamine and 25uM glutamate.

Plate cells 5 days or 12 days before transduction. Hippocampal neurons were plated at 5x104 cells/well and the cortical neurons were plated at 1x105

cells/well in 24 well poly-D-lysine coated plates. After four days, half of the media is removed and replaced with fresh NB/B27 media with L-glutamine but

no glutamate. Add 5ul and 10 ul (MOI depends on titer and cell density) of the concentrated lentivirus to each well. Do not add polybrene; it can be toxic to the cells.

Culture cells at 37oC for 6 hrs.

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If necessary, reapply the virus at the same concentrations to make the total in the 1st well 10ul and the 2nd well 20ul. (This step is necessary only if the viral titer is low and to maximize the number of transduced cells).

Culture overnight and next day replace media with fresh 0.5mL of media and culture three to four days. Assess expression by appropriate method (microscope for GFP, cell staining for lacZ)

Embryo transduction (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

According to Lois et al, 2002, Science 295:868-872, single cell mouse embryos can be transduced by Lentivirus. After 72 hrs in culture, transgene expression could be detected in the blastula- or morula-stage embryos that derive from the transduced zygotes.

According to Wolfgang et al, 2001, PNAS 98:10728-10732, day 7-8 blastocysts can also be transduced. Transduction in Chickens (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Lentiviral system has not been tested in chicken cells. However, a similar vector system has been used in developing chicken retinal cells: Refer to Coleman et al., Invest Ophthalmol. Vis. Sci. 43:1335-1343, 2002.

Most of our vectors contain the CMV promoter to drive transgenic expression and the CMV promoter has been shown to work effectively in chicken cells. Refer to Harvey et al, Nat Biotechnol.;20:396-399, 2002.

For chick embryo transduction, please refer to Pfeiffer et al, 2000, PNAS 97:12227-12232. Optimizing Lentiviral Transductions (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

Set up transductions in 96-well plates (or 24-well plates) in order to conserve virus. An easy way to optimize transduction is through the use of GFP lentivirus, although keep in mind that lentivirus containing certain genes of interest may need to be transduced at a higher MOI than GFP lentivirus in order to achieve optimal gene expression levels.

Use the table below for suggested MOIs and polycation concentrations on different cell types (or do a literature search if the cells of interest are not listed in the table). Polybrene is the most widely used polycation for lentivirus transductions; it enhances transduction efficiency in many cell types. If transducing a new cell line in cases where references on lentiviral transduction conditions are unavailable, first test cells for polybrene toxicity. Try several different concentrations of polybrene- 1.5, 3, or 6 ug/ml, as well as no polybrene.

Make the dilutions of polybrene in media and add 100 ul to cells at 30% confluency in a 96-well plate. Incubate for 24 hours and add fresh media. Examine cells for toxicity right after adding polybrene as well as 24 hours later. If toxicity is observed (compared to the un-treated cells) at all dilutions, then omit polybrene from the transductions. Some cells show toxicity only at the highest concentration.

An alternative to polybrene is DEAE-Dextran (6-10 ug/ml); this has been reported to work better than polybrene on certain cell types, such as B and T cells, but it may also be toxic to some cells, and the concentration may require optimization for use on the cell type of interest.

Transduction of adherent cells:

1) The day before transduction, plate cells such that they will be approximately 30% confluent the following day. Example: For 293 GripTiteTM cells, try plating 1.25 x 10e4 cells/well in 96-well plates (or 5x10e4 cells/well in a 24-well plate).

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2) The next day, prepare several virus dilutions in order to test a range of MOIs. Example: For HT1080 cells, try MOIs of 1, 5, and 10. For neural stem cells, try MOIs of 10, 25, 50, and 100. Also include polybrene (or DEAE-Dextran) at the appropriate concentration if desired (polybrene is toxic to some cell types, so it may need to be omitted as in the case of neural cells, or used at a reduced concentration – i.e. 3 ug/ml on 293 cells). DEAE-Dextran may also be toxic to some cell types. Note: Try to avoid using large volumes of virus. Virus should be kept to no more than 30% of the total volume in the well or maybe 50% at the very most; high-titer concentrated virus preps work best. The final volume in the wells (including virus plus media) should be 50-100 ul; 50 ul is preferable, but you can go up to 100 ul if you have a lower-titer virus. If using 24-well plates, the final volume of virus plus media should be about 250 ul. If toxicity is observed after transduction, decrease the virus volume (use a lower MOI) and/or omit polybrene.

3) Remove media and add the various virus dilutions to the cells. 4) Incubate overnight (about 24 hours), 37 degrees C. Remove virus and add fresh media. 5) Between 48 and 96 hours post-transduction, look for expression of GFP (or assay for expression of the gene of

interest). Note that some cell types, i.e. primary cells, it may take up to 3-4 days for maximal expression. In one rare case (human monocytes), maximal expression was not detected until 1 week post-transduction.

Transduction of suspension cells:

1) On the day of transduction, plate cells at a suitable density so that they will grow properly but will not need to be split for about 3-4 days. Plate cells in a small volume – i.e. 25-50 ul – in order to save room for virus.

2) Prepare virus dilutions to test different MOIs as described above. (Use polybrene or DEAE-Dextran at the appropriate concentration if desired). Aim for a final volume of 100 ul (virus plus media and cells). Add virus and pipet up and down at least 20 times with a P200 to make sure cells are in suspension. Place cells in the incubator. Mix cells every couple hours at least twice that day, and let transduction go overnight (about 24 hrs). Then add extra media – 50-100 ul (it is not essential to remove the virus). Proceed as above. Note: If using a very high MOI (i.e. 100) of CMV promoter-driven GFP lentivirus and no detectable expression is observed following transduction of the specific cell type, but good expression is detected in other cells (such as HT1080 or 293), consider trying a different promoter (such as EF1a, Ubc, a tissue-specific promoter, etc. with GFP in the MultiSite Gateway version of lentivirus). CMV is subject to silencing in some cell types.

Suggested range of lentiviral MOIs for particular cell types (based on transductions done with GFP lentivirus, by Invitrogen R&D, or in some cases, by customers using Invitrogen lentivirus):

Troubleshooting for Protein Expression (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures) If good transient expression is observed but no expression when titering viral particles, most likely the problem is at the packaging level. There are two possibilities- the lentiviral vector itself is not being packaged or it is packaged but not integrating.

If the LTRs are damaged. Test intactness of the LTRs by digestion of the pLenti vector with Afl II restriction enzyme. If the

requisite number of bands after Afl II digestion are not observed, then there is a problem with the LTRs and could explain the lack of gene transduction. Make sure that STBL3 cells are used for transformation.

Cells cell type/species suggested MOI range PolycationHT1080 human fibrosarcoma 1 to 10 6 ug/ml polybrene

293 human embryonic kidney 5 to 10 3 ug/ml polybreneHeLa human epithelial carcinoma 5 to 10 3 ug/ml polybrene

cortical neurons rat 10 to 25 noneneural stem cells rat 10 to 100 none

B cells mouse 10 to 35 6-10 ug/ml DEAE-DextranRAW 264.7 mouse macrophage 50 none

Mono Mac-6 human monocyte 100 none

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In all the experiments below, use the LacZ control plasmid in parallel (at digestion, and at packaging, and follow them up with beta-gal staining) or even better use the pLenti6.2 EmGFP plasmid since that will help pinpoint the exact problem.

If the gag/pol- or rev-expressing plasmids are suboptimal.

The presence of syncytia formation suggests that the pLP/VSVG vector is expressing the VSVG envelope protein as expected. Typically a lot of VSVG expression is not needed to induce syncitia formation; by contrast a lot of gag/pol is needed to form intact virion. If the problem is at the level of expression of gag/pol and/or rev, the amount of packaging mix can be increased as described in Tips to Improve Viral Titers.

Virus rescue (back to Table of Content) (back to Protocol and Application Notes) (back to Experimental Procedures)

This is a remote possibility if the stable cell line was to be transfected with the packaging mix. Although the promoter in the 5’LTR is inactive, this may happen if viral integration has occurred in a transcriptionally active locus and the transcription is driven from a cellular promoter. The safety feature included in our Lentiviral system will generally not allow this to happen. There is a splice donor and a splice acceptor site that removes the packaging signal (Psi) and inhibits viral production.

The splice donor and acceptor sites in the lentiviral vectors are derived from the HIV-1 genome. HIV-1 RNA undergoes extensive alternative splicing to generate various viral proteins. RNA splicing, as well as protease cleavage after protein synthesis, allows the virus to generate a lot of different proteins from a relatively small genome. A lot of other viruses also undergo alternative splicing - most likely, during their evolution, they “stole” splicing sites from their eukaryotic host cell genomes and incorporated them into their own genome, and they can take advantage of host cell splicing factors.

Safety Issues (back to Table of Content) (back to Protocol and Application Notes)

Key Safety Features of the ViraPower™ Lentiviral Expression System gp160 Envelope Protein Recombination or otherwise activation of endogenous retroviruses Activation of oncogenes Rev: splicing and safety in the ViraPower™ Lentiviral system Splicing and safety in the wild-type virus Risk Assessment Explanation of Biosafety Levels 1, 2, 3, 4 (BSL-1, -2, -3, -4)

Key Safety Features of the ViraPower™ Lentiviral Expression System (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues) In the US, the CDC suggests use of Lentiviral stocks to be treated as Biosafety Level 2 organisms*. For further information on BL-2 guidelines and lentivirus handling, please refer to: “Biosafety in Microbiological and Biomedical Laboratories”, 4th Ed. Centers for Disease Control and their website: www.cdc.gov. Invitrogen highly recommends users strictly follow all CDC published guidelines for BL- 2. There are many key safety features built into this 3rd generation HIV-based expression system to enhance its biosafety and minimize its relation to the wild-type human HIV-1 virus. It does however, contain vectors that encode for some HIV packaging proteins. Although it is up to each individual institution to determine if they want implement alternative environmental constraints, the ViraPower™ Lentiviral Expression System is far removed from the wild-type HIV virus on which it is based, and the following safety features and make this system safe for handling at BL-2.

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1) This is a four-plasmid system. The number of HIV-1 genes has been reduced to three (gag-pol and rev). These have been separated onto individual plasmids. The non-HIV envelope is on a third plasmid. These plasmids have been optimized in a packaging mix for easy use, and are included in the ViraPower™ Support Kit. All four plasmids have been engineered not to contain any regions of homology with each other to prevent unwanted recombination events that could lead to the generation of a replication competent virus. 2) The viral particles produced in this system are replication incompetent and only carry the gene(s) of interest. No other viral species are produced. This also means that none of the structural HIV genes (necessary for production of viral progeny) are present in the packaged viral genome. Gag, pol, rev and envelope genes are not present in the viral genome and are therefore never expressed in the target cell, so no new virus can be produced. 3) All four plasmids have been engineered to not contain any regions of homology with each other to prevent rare, but unwanted, recombination events that could lead to the generation of a replication competent virus. 4) The expression of the gag-pol sequence is rev-dependent, thus preventing the expression of gag-pol in the absence of rev. 5) The gene transfer vector pLenti6/V5 has been modified to be “self-inactivating” (Yu 1986, Yee 1987, Zufferey 1998). A deletion has been made in the 3’ LTR (called “delta U3”) that has no effect on the generation of viral genome for packaging in the producer cell. However once the produced virus transduces a target cell, the mechanisms of reverse transcription use the 3’LTR as a template to create the 5’LTR. The end result is an integrated viral genome that is defective in both its 5’ and 3’ LTRs. Thus, there is no transcription of the viral sequences in the target cell and no packagable viral genome is produced. This self-inactivation also allows long-term expression from adjacent heterologous promoters. This means that transduction with Invitrogen’s Lentiviral vectors ends with your gene of interest integrated into the host cell genome, for expression of your gene of interest with no production of viral particles. 6) Tat has been completely removed from our Lentivirus system. The HIV LTR (in the pLenti6 vectors) has been modified and is now a hybrid fusion between the RSV promoter and the 5' LTR, and this modification makes it a stronger promoter and removes its dependence on tat for virus production. Tat is not required in any downstream events (i.e. target cell transduction). 7) The stable cell lines that are made with the Lentiviral system would not contain any of the packaging proteins. These proteins are only used to package the viral particles from the 293FT cells. They are provided in trans, so they are not part of the viral particle themselves, and therefore not present when the target cell is transduced and the gene of interest stably integrated. Despite all of these safety features, the lentivirus produced with this system can still pose a biohazardous risk because it is fully capable of transducing primary human cells. For this reason, Invitrogen and Cell Genesys treat these viruses as Biosafety Level 2 organisms and strictly follow all CDC guidelines for BL-2. Extra care should be taken when creating viruses carrying harmful or toxic genes (such as activated oncogenes). For further information on BL-2 guidelines and lentivirus handling, please refer to: “Biosafety in Microbiological and Biomedical Laboratories”, 4th Ed. Centers for Disease Control and their website: www.cdc.gov. gp160 Envelope Protein (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues)

There is a region of overlap after alignment of the gp160 DNA sequence with pLenti6/V5-D-TOPO; however this overlap region does not encode gp160. The first 400 bases of gp160 are deleted in this region. The region of envelope left in the vector lacks an initiator ATG as well as the first 133 amino acids. Any transcript that is produced in the packaging transfection will still lack a functional envelope.

Since the lentiviral vector has a self-inactivating feature, there is a minimal chance of having promoter driven expression of the truncated env fragment left in the vector. The SIN feature ensures that the

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enhancer region in the 3' LTR does not get copied into the 5' LTR to drive lentiviral genome expression. Even if the virus integrates into a transcriptional hot spot, the 400 bp deletion in envelope assures that functional env doesn't get expressed. The env-deleted region is also upstream of the CMV promoter so the internal promoter does not drive expression of the deleted region.

A portion of gp160 is left in the vector to provide functionality and safety. The part of the envelope region left in the vector comprises the RRE feature. This region ensures that the viral genome gets transported from the nucleus to the cytoplasm to be packaged into virions. Thus, this region of env must be in the transfer vector. The RRE is functional in the presence of the Rev protein. The safety feature here resides in the fact that Rev is provided on another plasmid, pLP2, to minimize against recombination.

Since there is no Gag, pol, rev or functional env genes present in the lentiviral vector, these proteins are not expressed in the target cell.

Recombination or otherwise activation of endogenous retroviruses (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues) It is certainly possible that a recombination event could occur with endogenous retroviruses (this would be true for any retroviral delivery system), however there are no HIV accessory genes in the lentiviral system, so there would not be any chance for generating wt HIV. If working with cells already containing integrated HIV, the recombination chance would be higher, but in this case strict BL2 safety guidelines should be followed. Activation of oncogenes (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues) It has been documented that any retrovirus can integrate somewhere in the genome that causes activation of an oncogene. This is not unique to lentivirus, although our vectors are safer due to the inactivation of both LTRs upon integration into the genome. Rev: splicing and safety in the ViraPower™ Lentiviral system (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues)

The key to HIV-1 splicing is the Rev protein (supplied in our pLP2 vector). When one co-transfects the packaging mix (containing pLP2) into 293FT cells, Rev accumulates and stimulates the nuclear export of unspliced and singly spliced RNA into the cytoplasm for encapsidation. Unspliced RNA becomes the viral genome after encapsidation.

After transduction into the target cell, rev is no longer needed because transcription of the gene of interest will proceed from the internal promoter (say CMV), and expression of the unspliced proviral genome is not needed (this wouldn't happen anyway because of the self-inactivation feature in our vectors).

The cellular triggers for splicing reside in the spliceosomes/hnRNPs that Rev interacts with. For a reference see Verhoef et al, (2001) Repair of a Rev-Minus Human Immunodeficiency Virus Type 1 Mutant by Activation of a Cryptic Splice Site. Journal of Virology, 75:3495-3500, 2001.

Co-transfecting Rev together with the pLenti vector, will result in a high level of expression of Rev. This suppresses any splicing events that would otherwise remove psi and RRE. This is important because if psi was removed in 293FT, empty virions will be created lacking any genome carrying the gene of interest.

If the RRE is deleted, there will be no transport of the transcript from the nucleus to the cytoplasm for packaging (i.e. Rev must be present for this putative viral transcript to make it out of the nucleus; no Rev, no viral genome).

In the absence of the pLP2 vector (i.e. in the absence of Rev) one would get mostly multi-spliced RNA and the viral titers would drop significantly. Relatively lower titers may also be found if insufficient amounts of Rev were present.

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In the ViraPower™ Lentivirus System, once the proviral DNA is integrated into the transduced cell, the psi signal becomes irrelevant because the SIN feature ensures that the entire viral genome cannot get transcribed. Even if the genome were to get transcribed somehow, the presence of RRE will require that Rev be supplied in order to get the RNA out of the nucleus to become a viral genome.

Since Rev is supplied in trans in only the 293FT cell line and is absent in the target cell, the chances of creating an infectious virus after transducing the pLenti becomes even more remote. This is the safety feature built into the RRE/Rev elements of the Lenti vectors. This safety feature is witnessed in the transduced cell, but the groundwork for that safety feature begins in 293FT.

Splicing and safety in the wild-type virus (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues)

Immediately after infection and integration, transcription of the entire viral genome occurs from the promoter elements in the U3 region of the 5'LTR. Since initially there is little or no Rev protein at the beginning of infection, multiply spliced RNA is produced that lead to the expression of Tat, Rev, and Nef. Since the packaging signal is spliced out, and the RRE is removed, these multiply spliced RNAs don't get packaged or express envelope protein.

When rev accumulates (from the multiply spliced), it changes the expression profile from multiply spliced to unspliced and singly spliced RNA.

Since the unspliced RNA contains the Rev Responsive Element, RRE, nuclear export of this RNA depends on the accumulation of Rev. Due to the presence of the packaging signal, psi, the unspliced RNA can now be packaged into the virion to serve as the viral genome.

This results in an infectious virus that can infect a host cell and the cycle repeats itself. Risk Assessment (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues) The following risk assessment assesses the risk to human health and environment as minimal, with a final ACGM Classification as Class I. This pertains only to the shipping and storage of these products, and the handling of this product prior to it's being packaged in viral capsid form. PRODUCT(S): ViraPower™ Lentiviral Directional TOPO® Expression Kit K4950-00 ViraPower™ Lentiviral Gateway™ Expression Kit K4960-00 pLenti6/V5 Directional TOPO® Cloning Kit K4955-10 pLenti6/V5-DEST™ Gateway™ Vector Pack V496-10 ViraPower™ Lentiviral Support Kit V4970-00 293FT Cell Line R700-07 ViraPower Zeo Lentiviral Gateway Expression kit K4980-00 ViraPower Ubc Lentiviral Gateway Expression Kit K4990-00 ViraPower Promoterless Lentiviral Gateway Kit K5910-00 ViraPower T-REx Lentiviral Expression System K4965-00 BLOCK-iT™ Lentiviral RNAi Expression System K4944-00 BLOCK-iT™ Inducible H1 Lentiviral RNAi System K4925-00 Block-iT Lentiviral Pol II miR RNAi Expression System K4937-00 Block-iT Lentiviral Pol II miR RNAi Expression System with EmGFP K4938-00 ViraPower II Lentiviral Gateway Expression Kit K367-20 ViraPower II Lentiviral C-Lumio Gateway Expression Kit K370-01 ViraPower II Lentiviral N-Lumio Gateway Expression Kit K371-20 Vivid Colors pLenti6.2-GW/EmGFP V369-20 BRIEF DESCRIPTION:

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The ViraPowerTM lentiviral expression systems allow creation of a replication-incompetent, HIV-1 based lentivirus which can be used to deliver and express a gene of interest in either dividing or non-dividing mammalian cells. ASSESSMENT OF RISK TO HUMAN HEALTH: Minimal, since kits made up of several separate components, no one of which can produce viable virus particles. In addition all kit components stored at low temperatures and are therefore biologically inactive and can only be kept alive at higher temperatures if given the appropriate specialized culture conditions. ASSESSMENT OF RISK TO THE ENVIRONMENT: Minimal, since kits made up of several separate components, no one of which can produce viable virus particles. In addition all kit components stored at low temperatures and are therefore biologically inactive and can only be kept alive at higher temperatures if given the appropriate specialized culture conditions. FINAL ACGM CLASSIFICATION: CLASS 1 ACTIVITY CAUTION: This assessment covers the shipping and storage of these products only! When these products are used in the laboratory it is strongly recommended that class 2 conditions or greater be practiced and it is the responsibility of the customer to ensure that this product is used in accordance with all applicable local legislation and guidelines, including EC Directive 90/219/EEC on the contained use of genetically modified organisms. ViraPower™™ Lentiviral Expression System Manual (Version A 022802, 25-0501) RISK GROUP - Details are on pages 36 and 40 of the CDC Guidelines: The wild type HIV-1 virus is infectious, can cause disease and has preventive or therapeutic intervention available. But the lentiviral vector is NOT infectious in the sense that it cannot produce infectious progeny. Additionally the vector has only 25% of the virus genomes left and has multiple safety features built-in. Therefore the vector system itself is safe. However it is in Risk Group 3 because it is based upon HIV-1. Explanation of BioSafety Level 1, 2, 3, 4 (BSL-1 -2 -3 -4) (back to Table of Content) (back to Protocol and Application Notes) (back to Safety Issues) Follow this link to the online Laboratory Biosafety Guidelines 2nd Edition 1996, and 3rd edition 2004. This guidebook contains explanations of what organisms must be treated at what safety levels and what the physical requirements are for a facility to meet those safety levels. http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl-96/ PRODUCT DOCUMENTATION (back to Table of Content)

Brochures Cell lines Citations

COA FAQ Licensing

Manuals MSDS Newsletters

Vector Data

REFERENCES (back to Table of Content)

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Books and Review articles about retrovirus

1. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=rv.TOC 2. Buchschacher G.L. and F. Wong-Staal , Blood, Vol. 95, issue 8, p. 2499-2504, Development of lentiviral

vectors for gene therapy for human diseases

References citing use of Invitrogen ViraPower™ system 1. Rubinson DA et al. A lentivirus-based system to functionally silence genes in primary mammalian cells,

stem cells and transgenic mice by RNA interference. Nat Genet 2003 Mar;33(3):401-6 2. Espenshade et al. Proc. Natl. Acad. Sci. USA 2002; 99:11694; Note: The lentivirus enters a cell in a

receptor-independent manner that could involve endocytosis 3. Aiken (1997) J. Virol. 71:5871. Note: same as above. (not using the ViraPower™ system, but refers to

use of VSV-G). 4. Kevin C. Wang, Jieun A. Kim, Rajeev Sivasankaran, Rosalind Segal & Zhigang He NATURE | 20

OCTOBER 2002 | doi:10.1038/nature01176 |www.nature.com/nature © 2002 (transfection of neurons) 5. Lois et. al. Germline transmission and tissue-specific expression of transgenes delivered by Lentiviral

vectors. Science. 2002 Feb 1;295(5556):868-872. 6. Hikida et al. Coupling Between B Cell Receptor and Phospholopase C-{gamma}2 Is Essential for Mature

B Cell Development. J. Exp. Med. (2003) 198: 581-589. 7. Masaki Mogi, Jiang Yang, Jean-Francois Lambert, Gerald A. Colvin, Ichiro Shiojima, Carsten Skurk,

Ross Summer, Alan Fine, Peter J. Quesenberry, and Kenneth Walsh Akt Signaling Regulates Side Population Cell Phenotype via Bcrp1 Translocation J. Biol. Chem., Oct 2003; 278: 39068 - 39075.

8. Makoto Murakami, Karin Nakashima, Daisuke Kamei, Seiko Masuda, Yukio Ishikawa, Toshiharu Ishii, Yoshihiro Ohmiya, Kikuko Watanabe, and Ichiro Kudo Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and –2 J. Biol. Chem., Sep 2003; 278: 37937 - 37947.

References citing the use of lentiviral vectors (non Invitrogen vectors)

1. Dull, et. al. (1998) J. Virology, 72:8463-8471. 2. Lois, et. al. (2002) Science, 295:868-872. (RNAi ref.) 3. Mitta, et. al. (2002) Nucleic Acid Res., 30:e113. 4. Cui, et. al. (2002) Blood, 99:399-407. 5. Miyoshi, et. al. (1997) Proc. Natl. Acad. Sci. USA, 94:10319-10323. 6. Kafri, et. al. (1997) Nat. Genet., 17:314-317. 7. Wu, et. al. (2000) Mol. Therapy, 2:47-55. 8. Zufferey, et. al. (1998) J. Virol., 72:9873-9880. 9. Lewis, et. al. (1994) J. Virol., 68:510-516. 10. Baek, et. al. (2001) Hum. Gene Ther., 12:1551-1558. 11. Pfeifer, et. al. (2002) Proc. Natl. Acad. Sci. USA, 98:11450-11455. (RNAi ref.) 12. Mochizuki, et. al. (1998) J. Virol., 72:8873-8883. 13. Bovia, et. al. (2003) Blood, epub ahead of print 14. Ma, et. al. (2003) Stem Cells, 21:111-117. 15. Wang et. al. (2003) Nature, 420(7):74-78 16. Qin et. al. (2003)* PNAS, 100(1): 183-188 17. Rouas et. al. (2002) Cancer Gene Therapy, 9:715-724 18. Cavalieri et. al. (2003) Blood (3) 813-822 19. Kamei et. al. (March 7, 2003) JBC online. 20. PNAS Vol. 93, Issue 21, 11382-11388, October 15, 1996

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector

21. Mol Ther. 2002 Apr;5(4):397-404. In vivo gene delivery to synovium by lentiviral vectors. (Injection into rat knee joints)

24. Gene Therapy (2003) 10, 1917–1925 Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction

25. Lentivirus Gene Engineering Protocols. Federico, Maurizio (Istituto Superiori di Sanità, Rome, Italy)

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Following chapters available as promotional items from Invitrogen thru July 2005: Chapter 6: “Detection and Selection of Lentiviral Vector-Transduced Cells,” by Yan et al. As part of

the Methods in Molecular Biology series, learn how HIV-1-based lentiviral vectors show high-transduction efficiencies for many human and animal cell types.

Chapter 18: “HIV-2 Vectors,” by Arya et al. Learn practical methods of lentivirus-based gene transfer into stem cells.

PRODUCT NAME AND CATALOG NUMBERS (back to Table of Content)

Name Catalog Number ViraPower™ Lentiviral Gateway Expression Kit K496000 pLenti6/V5-DEST Gateway Vector V49610 ViraPower™ Zeo Lentiviral Gateway Expression Kit K498000 pLenti4/V5-DEST Gateway Vector V49810 ViraPower™ UbC Lentiviral Gateway Expression Kit K499010 pLenti6/UbC/V5-DEST Gateway Vector V49910 ViraPower™ Lentiviral Directional TOPO Expression Kit K495000 pLenti6/V5 Directional TOPO Cloning Kit K495510 ViraPower™ Zeo Lentiviral Support Kit K498500 ViraPower™ Bsd Lentiviral Support Kit K497000 ViraPower™ Lentiviral Packaging Mix K497500 ViraPower™ T-REx Lentiviral Expression System K496500 ViraPower™ T-REx Lentiviral Gateway Vector Kit K496700 pLenti6.2-GW/EmGFP Expression Control Vector V36920 ViraPower II Lentiviral Gateway Expression System K36720 pLenti6.2/V5-DEST Gateway Vector V36820 ViraPower™ II Lentiviral C-Lumio Gateway Expression System K37020 ViraPower™ II Lentiviral N-Lumio Gateway Expression System K37120 BLOCK-iT™ Lentiviral RNAi Expression System K494400 BLOCK-iT™ Lentiviral RNAi Gateway Vector Kit K494300 BLOCK-iT™ Inducible H1 Lentiviral RNAi System K492500 BLOCK-iT™ Lentiviral RNAi Zeo Gateway Vector Kit V48820 Block-iT™ Lentiviral miR RNAi Expression System K493700 Block-iT™ Lentiviral miR RNAi Expression System with EmGFP

K493800

ViraPower™ Promoterless Lentiviral Gateway Expression Kit With MultiSite Gateway Technology

K591000

ViraPower™ Promoterless Lentiviral Gateway Vector Kit With MultiSite Gateway Technology

K59110

pLenti-bsd/irf1-bla Vector K1209 pLenti-bsd/DBE-bla Vector K1210 pLenti-bsd/ARE-bla Vector K1211

COMPONENTS (back to Table of Content) Systems include

Expression plasmid that allows packaging into viral particles and expression of gene from strong CMV promoter or promoter of choice (promoterless)

A control expression plasmid containing the lacZ gene which, when packaged into virions and transduced into a mammalian cell line, expresses Beta-galactosidase.

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An optimized mix of the three packaging plasmids (pLP1, pLP2, and pLP/VSVG) which supply the structural and replication proteins in trans that are required to produce the lentivirus

Expression kits contain 3 manuals (system manual, cell manual, vector manual) ViraPower™ Lentiviral Directional TOPO Expression System (K495000)

Name Size Part Number pLenti6/V5-Directional TOPO vector 20 reactions 450238 ViraPower™ Lentiviral Support kit NA 440190 293FT cells 3 x 106 cells in 1 ml 510035 Blasticidin 50 mg 461120 One-shot Stbl3 E. coli 20 x 50 ul C737303

ViraPower™ Lentiviral Gateway Expression System (K496000)

Name Size Part Number pLenti6/V5-DEST vector 6 ug 430315 ViraPower™ Lentiviral Support kit NA 440190

293FT cells 3 x 106 cells in 1 ml 510035

Blasticidin 50 mg 461120

One-shot Stbl3 E. coli 20 x 50 ul C737303

ViraPower™ Zeo Lentiviral Gateway Expression System (K498000) Name Size Part Number pLenti4/V5-DEST vector 6 ug 430130 ViraPower™ Zeo Lentiviral Support kit NA 442010 293FT cells 3 x 106 cells in 1 ml 510035 One-shot Stbl3 E. coli 20 x 50 ul C737303

ViraPower™ UbC Lentiviral Gateway Expression System (K499000)

Name Size Part Number pLenti6/UbC/V5-DEST vector 6 ug 430131 ViraPower™ Lentiviral Support kit NA 440190 293FT cells 3 x 106 cells in 1 ml 510035 Blasticidin 50 mg 461120 One-shot Stbl3 E. coli 20 x 50 ul C737303

ViraPower™ Zeo Lentiviral Support Kit (K498000, and part 442010)

Name Size Part Number ViraPower™ packaging mix 195 ug 351275 Lipofectamine 2000 0.75 ml 52758 Zeocin 1.5 ml 460509

ViraPower™ T-REx Lentiviral Expression System (K496500)

Name Size Part Number ViraPower™ T-REx Lentiviral vectors and Tertracycline

NA 431616

LR Clonase Enzyme II NA 11791020 ViraPower™ Lentiviral Support kit NA 440190 293FT cells 3 x 106 cells in 1 ml 510035 Blasticidin 50 mg 461120 One-shot Stbl3 E. coli 20 x 50 ul C737303

ViraPower™ T-REx Lentiviral Gateway Vector Kit (K496700, and part 431616)

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Name Size Part Number pLenti6/TR 20ug 352005 pLenti4/TO/V5-DEST 6 ug 352007 pLenti4/TO/V5-GW/LacZ 10 ug 352009 Tetracycline 10 mg/ml in water 471112

ViraPower™ Promoterless Lentiviral Gateway Expression Kit With MultiSite Gateway Technology (K591000)

Name Size Part Number pENTR5’-TOPO TA Cloning Kit 20 reactions 450711 pLENTI6/R4R2/V5-DEST vector Kit NA 450712 LR Clonase Plus Enzyme Mix NA 12538013 ViraPower™ Lentiviral Support kit NA 440190 293FT cells 3 x 106 cells in 1 ml 510035 Blasticidin 50 mg 461120 One-shot Stbl3 E. coli 20 x 50 ul C737303

ViraPower™ Promoterless Lentiviral Gateway Vector Kit With MultiSite Gateway Technology (K59110)

Name Size Part Number pENTR5’-TOPO TA Cloning Kit 20 reactions 450711 pLENTI6/R4R2/V5-DEST vector Kit NA 450712 One-shot Stbl3 E. coli 20 x 50 ul C737303

pLenti6/R4R2/V5-DEST vector kit (part 450712)

Name Size Part Number pLENTI6/R4R2/V5-DEST vector 6 ug 461021 pENTR5’/UbCp 10 ug 461022 pLenti6/UbC/V5-GW/lacZ 10 ug 350857

ViraPower II Lentiviral Gateway Expression System (K36720)

Name Size Part Number pLenti6.2/V5-DEST vector 6 ug 431904 ViraPower™ Lentiviral Support kit NA 440190

293FT cells 3 x 106 cells in 1 ml 510035

Blasticidin 50 mg 461120

One-shot Stbl3 E. coli 20 x 50 ul C737303

pLenti6.2-GW/EmGFP Expression Control Vector (V36920) Name Size Part Number pLenti6.2-GW/EmGFP Expression Control Vector

20 ug 431907

ViraPower™ II Lentiviral C-Lumio™ Gateway Expression System (K37020)

Name Size Part Number pLenti6.2/C-Lumio™/V5-DEST

6 ug 431905

pLenti6.2/ C-Lumio™/V5-GW/lacZ control 10 ug 431905 ViraPower™ Lentiviral Support kit NA 440190 293FT cells 3 x 106 cells in 1 ml 510035 Blasticidin 50 mg 461120 One-shot Stbl3 E. coli 20 x 50 ul C737303

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Lumio™ Green In-Cell Labeling Kit

2 mM in DMSO (53.2 ug in 40 ul) MW: (664.50 g/mol)

457510

ViraPower™ II Lentiviral N-Lumio™ Gateway®Expression System (K37120)

Name Size Part Number pLenti6.2/N-Lumio™/V5-DEST

6 ug 431906

pLenti6.2/ N-Lumio™/V5-GW/lacZ control 10 ug 431906 ViraPower™ Lentiviral Support kit NA 440190 293FT cells 3 x 106 cells in 1 ml 510035 Blasticidin 50 mg 461120 One-shot Stbl3 E. coli 20 x 50 ul C737303 Lumio™ Green In-Cell Labeling Kit

2 mM in DMSO (53.2 ug in 40 ul) MW: (664.50 g/mol)

457510

ASSOCIATED PRODUCTS (back to Table of Content)

Name Size Catalog Number

One Shot Stbl3 Competent Cells

20 reactions C737303

Library Efficiency DB3.1 Competent cells

1 m ( 5 x 0.2 ml) 11782018

293FT Cell Line 20 reactions R70007 Gateway LR Clonase II Enzyme Mix

20 reactions 11791020

100 reactions 11791100 pLenti6/TR vector kit 20 ug V48020 ViraPower™ Packaging Mix 60 rxns K497500 Lipofectamine 2000 0.75 ml 11668027 1.5 ml 11668019 Geneticin 1 gm 11811023 5 gm 11811031 Zeocin 1 gm R25001 5 gm R25005 Blasticidin 50 mg R21001 Beta-gal antiserum 50 ul R90125 Beta-gal Assay Kit 100 reactions K145501

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