The following protocol for cloning of gene trap cassettes into proviral vectors,

retrovirus production and infection was written by Tetsuya S. Tanaka, Ph.D. at the

Stanford Lab, based on references (1-6). All of the standard procedures for molecular

cloning techniques will be found in “Molecular Cloning, 3rd ed.”, unless otherwise noted.

1. Cloning of gene trap cassettes into proviral vectors.

1.1 Comments about cloning into proviral vectors.

The self-inactivating (SIN) proviral vectors, pGen- (4) and its derivative pGep-, in

which cryptic splice sites found in pGen- were removed (1), were originally developed

and kindly provided by Dr. Phill Soriano at Fred Huchinson Cancer Center, WA, USA.

Gene trap cassettes will be inserted into reverse orientation relative to viral transcription

(2). The vectors should be transformed into E. coli strain HB101 to minimize undesired

recombination events (as described in Ref. 4, pGen- was designed with low-copy-number

pBR322 as a backbone to prevent recombination. So any precaution to reduce its copy

numbers and activities of DNA recombinases would, I think, help to minimize the

frequency of recombination events). To clone gene trap cassettes, pGen- has only XhoI

site as an unique cloning site, whereas pGep- has ClaI, SalI and XhoI sites as unique

cloning sites (Fig.1). By standard linker ligation method, these unique restriction enzyme

sites can be introduced at both ends of cassettes. No alkaline phosphatase treatment of

linearlized pGen- or pGep- gives better cloning efficiency.

1.2 Ligation.

1. Mix DNAs of the vector and a gene trap cassette at 1:3 ratio and ligated with a ligation

kit (Roche and New England Biolabs provide similar quick ligation kits; NEB’s kit is

simpler and has better efficiency). No alkaline phosphatase treatment of vector DNAs is

required.

2. After transformation of the ligation products into E. coli HB101 with a standard

method, plate cells onto LB/Amp plates and incubate at RT for 2-3 days. If the cloning

is successful, several hundreds of ampicillin-resistant bacterial colonies can be

obtained, whereas even without any alkaline phosphatase treatment, almost no colony

derived from self-ligation appears.

3. To minimize selection of negative, background clones that have no inserts (see Fig. 3),

colonies should be screened by colony-lift, after re-arrayed onto new LB/Amp plates

and grown at RT for 2-3 days (or directly), instead of carrying out direct mini-preps of

plasmid DNA or direct PCR.

1.3 Colony Hybridization (more detailed procedures will be found in “Molecular

Cloning, 3rd ed.”).

1. Soak blotting papers (VWR, #28298-020) in 1) 10% SDS, 2) denaturing buffer (1.5M

NaCl, 0.5M NaOH), and 3) neutralization buffer (1.5M NaCl, 1M TrisHCl, pH7.4)

individually and place them on different trays. Remove excess solutions.

2. Place sterile nylon membranes (Hybond-N+; Amersham Biosciences, #RPN303B) on

top of the bacterial colonies for a few minutes, and with the side of bacterial colonies

up, transfer them into “10% SDS”, “denaturing buffer”, “neutralization buffer” serially

in this order for 5 min each.

3. Wash the neutralized membranes with 2x SSC for 5min, and then treat them with

2mg/ml Proteinase K (Roche, #3115828) in 2x SSC at RT for 40min to remove

bacterial debris, which cause high background hybridization signals in many occasions.

4. Wash the membranes with 2x SSC briefly, cross-link with UV at 1200 x 100 J/cm2,

and dry them in between dry blotting papers. The dried membranes can be stored at RT

for several days.

5. Wash the membranes with 2x SSC. Any residual bacterial debris can be wiped with

wet Kim-wipes in 2x SSC.

6. Prehybridize the membranes with QuickHyb solution (Stratagene, #201220)

supplemented with heat-denatured Salmon sperm DNA (Invitrogen, #15632-011; for

10ml hyb solution, 100µl of 10mg/ml Salmon sperm DNA is required) at 65°C for

1hr.

7. Radio-label template DNA as a probe with RadPrime DNA labeling system

(Invitrogen, #18428-011). No purification of probe is required without getting any high

background.

8. Add heat-denatured, labeled probe into prehybridization solution, mix well and

hybridize at 65°C for 2hr.

9. Wash the hybridized membranes as follows; 2 washes with 2x SSC, 0.1% SDS at RT

for 30min each, 2 washes with 2x SSC, 0.1% SDS at 65°C for 30min each, and 2

washes with 0.1x SSC, 1% SDS at 65°C for 30min each. Typically activities on

membranes are still very high even after the highest stringent washes.

10. Expose the membranes onto an x-ray film (Biomax; Kodak, #829-4985) in a cassette

at -86°C for overnight. Often shorter exposure (30min and/or 2hr) is required

depending on activities on membranes.

11. Develop the film (Fig. 2).

12. Based on the hybridization results, positive bacterial colonies are further examined by

purifying plasmid DNAs followed by restriction enzyme mapping to identify the clones

with correct size and orientation of the insert (Fig. 3). For example, one clone out of

about 200 colonies screened was the one with a gene trap cassette in correct orientation.

Once positive, correct clones are identified, they can be grown at 37°C without any

problem.

2. Production of retrovirus.

2.1 Culture of PhoenixTM cells.

PhoenixTM cells (3, 6) were cultured in Dulbecco’s modified Eagle medium

(DMEM; Gibco, #11960-044) supplemented with 10% fetal bovine serum (Gemini Bio

Products, #100-106), 1x Penicillin/Streptomycin (Sigma, P7539), 1x GlutaMAX (Gibco,

#35050-061) at 37°C, 5% CO2. Every two days at 70-80% confluency, trypsinized

(Tryple Express; Gibco #12605-010) Phoenix TM cells were passaged at 1:5.

Every once in a few months of culture, it is recommended to select Phoenix TM

cells with 300µg/ml Hygromycin B and 1µg/ml Diphtheria Toxin for one week to

maintain their high-titer virus production. Then large number of stocks at early passages

should be prepared.

2.2 Collection of culture supernatant containing retroviruses by transient

transfection of proviral vectors into Phoenix TM cells (3, 5).

1. Prepare 2M CaCl2 and 2x Hepes-buffered saline (2x HeBS; 280mM NaCl, 10mM KCl,

1.5mM Na2HPO4, 12mM (d)-glucose, 50mM HEPES, pH7.00 adjusted with 0.5N

NaOH) in cell-culture grade H2O and filtrate them with 0.22µm filter (Millex -GP;

Millipore, SLGP033 RS). Make aliquots into 1ml each for 2M CaCl2 and 5ml each for

2x HeBS, and store them at -20°C.

2. The day before transfection, plate 2-3 x 106 Phoenix TM cells in a 10-cm dish and

culture for overnight.

3. Mix 30-40 µg of supercoiled proviral DNA in 438µl H2O, with 62µl of 2M CaCl2 in a

14ml Falcon Round-Bottom Tube (#35-2059) per transfection.

4. While vortexing the tube vigorously, add 500µl 2x HeBS drop-wise with 1ml pipette.

Mix very well (bubbles can be formed)!

5. While swirling the 10-cm dish gently, add the DNA/CaCl2 mixture into culture

medium drop-wise and incubate at 37°C, 5% CO2 for overnight (14-17 hr).

Precipitates of DNA should be visible under a microscope. To measure the efficiency

of transfection, any expression construct with GFP can be used in parallel (Fig.4),

although the size of proviral DNA would be much larger, so that the efficiency of

transfection for proviral DNA would be less with the same amount of DNA used.

Figure 4. Typical results of transient transfection of a GFP construct with CaPO4into Phoenix TM cells. Expression of GFP is visible in 80-90% of cells in ~24hrpost transfection.

6. In 14-17hr post transfection, feed the cells with 6-10ml of fresh medium for virus

production and culture them at 37°C, 5% CO2 for 2-3 days. Culturing transfected cells

at 30°C may help to reduce degradation of viruses whereas production of virus is

slow.

7. Collect culture supernatant, filtrate it with 0.22µm filter (Millex -GP; Millipore,

SLGP033 RS) to remove cellular debris, make aliquots into 1ml each and store at

-80°C until used. Do not repeat freezing and thawing of the culture supernatant.

Because ecotropic viruses produced as above are relatively unstable, keeping culture

supernatant at lower temperature will help to keep the higher titer of virus.

8. Cell lines producing viruses stably can be established with Phoenix TM cells as

described in Ref. 4. The cell lines with higher virus titer will be identified by dot-

blotting.

3. Infection of retroviruses into ES cells

1. The day before infection, plate 3x 106 R1 ES cells in 10-cm dishes with ES culture

medium with LIF.

2. Thaw the culture supernatant at RT and dilute it into 1:10-15 with ES culture medium

with LIF, and 4µg/ml polybrene (Hexadimethrine bromide; Sigma H9268-5G) to

enhance the single infection of virus.

3. Remove ES culture medium in dishes, add 3-4ml of the virus mixture onto ES cells

and incubate them on a rocker platform at 37°C, 5% CO2 for 3-5hrs.

4. Add 6-7ml more ES culture medium with LIF and 4µg/ml polybrene and incubate

them further for ~20hrs.

5. Start drug selection of infected ES cells. In case of G418 selection (Geneticin; Gibco,

#10131-035; 167µg/ml in ES culture medium with LIF), resistant colonies will appear

in 10 to 14 days. Typically about 15 to 60 of G418-resistant colonies per plate were

obtained. Based on Southern blot analysis, less than one out of 96 (1%) had multiple

insertion of viruses into genome with this protocol.

6. Isolate G418-resistant colonies into 96-well plates.

References

1. Chen W.V., et al. (2004) Identification and validation of PDGF transcriptional targets

by microarray-coupled gene-trap mutagenesis. Nat. Genet. 36, 304-312.

2. Friedrich G. and Soriano P. (1991) Promoter traps in embryonic stem cells: a genetic

screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513-1523.

3. Pear W.S., et al. (1993) Production of high-titer helper-free retroviruses by transient

transfection. Proc. Natl. Acad. Sci. USA 90, 8392-8396.

4. Soriano P. et al. (1991) Promoter interactions in retrovirus vectors introduced into

fibroblasts and embryonic stem cells. J. Virol. 65, 2314-2319.

5. Yao S, et al. (2003) Retrovirus silencer blocking by the cHS4 insulator is CTCF

independent. Nuc. Acid Res. 31, 5317-5323.

6. Nolan Lab home page:

http://www.stanford.edu/group/nolan/retroviral_systems/retsys.html