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Protocols

Long DNA oligos containing the target sequence are cloned into LentiLox 3.7. Once clones have been isolated, virus is produced by transfecting 293 cells and collecting supernatant. This supernatant is then used to infect cells of interest directly, or concentrated for use in embryo infections. Note: These lentiviruses are pseudo-coated with VSV-G and are capable of infecting human cells, and thus present important biosafety issues.

Virus Production

1. Plate 12 x 10⁶ 293.T in 20 ml on a 15 cm² plate 24 hours before transfection. In general, two 15cm plates per virus. It is essential that the cells be well-maintained and of relatively low passage number.
2. Mix the following DNAs (made w/ Endo-free Qiagen Kits) in a FACS tube. The DNAs should be in Endo-free TE at a concentration of 0.5mg/ml.

For 3 plasmid system:

20 ug vector,

10 ug VSVG

15 ug D 8.9

For 4 plasmid, system (recommended),

20 ug vector,

10 ug VSVG

10 ug RSV-REV

10 ug pMDL g/p RRE

3. Add 400 ul 1.25 M CaCl₂ and 1.5 ml H₂0 and mix by tapping gently.

The following steps are done 1 plate at a time.

4. Add 2 ml of 2X HBS dropwise to DNA mixture while bubbling with a Pasteur pipette. When finished, continue to bubble for 12-15 seconds.
5. Take plate of 293T out of the incubator (plate remains in incubator for long as possible), and add transfection mixture dropwise all over the plate. Gently swirl plate from front to back, and return immediately to incubator.
6. 3.5 to 4 hours later, remove media, wash 2x with 10ml warm PBS, and add 20 ml warm D10 onto plate and place in incubator.
7. 36-48 hours after transfection, harvest viral supernatant and spin @ 2000 rpm, 7 min at 4C in a 50ml tube.
8. Filter viral SN through .45 um filter. Add 35ml of filtered supernatant to an ultracentrifuge tube. Balance tubes with additional media. Cover tubes with small piece of parafilm. (It is useful to titer some of the leftover supernatant to determine if there is loss of virus during concentration.)
9. Spin tubes using a SW-28 rotor @ 25,000 rpm, 90 min, 4C. Decant liquid and leave tube upside down on kimwipe for 10 min. Aspirate remaining media being careful not to touch bottom of tube.
10. Add 15ul cold PBS (for embryo infections, or any volume you wish) and leave tube at 4C O/N with no shaking.
11. To resuspend, hold tube at angle and pipet fluid over pellet 20 times, being careful not to touch pellet with tip. It is expected that the pellet not be resuspended after this is complete. This pellet does not contain virus and can be discarded.
12. Aliquot or use virus. Virus should be aliquoted, flash-frozen in liquid nitrogen and stored at -80C. There should be no change in titer with freezing concentrated virus. Avoid multiple freeze-thaws.

1. Plate 4x10⁵ 293.T cells/well in a 6-well plate 12-24 hours prior to titering. It is helpful to have an additional well as a negative control that you mock infect with D10+polybrene but without virus.

2. Make a stock solution of D10 with 8ug/ml polybrene.

3. Generate a 10-fold dilution series of virus in the D10+polybrene. Using 1.5mls/well you should have 1ul , .1, .01, .001, .0001, and .00001uL of virus/well.

4. Incubate at 37 degrees O/N. Replace media with fresh D10.

5. At least 48 hours after infection trypsinize cells for FACS analysis. (Trypsinize, inactivate with media, spin, and resuspend in cold PBS).

6. FACS analyze for EGFP expression and record the percentage of cells that are EGFP positive.

7. Use a well that has between .1% and 10% of cells expressing EGFP to determine titer.

Sample calculation assuming 1% infection from the well with .01ul of virus.

.01 (percentage of cells that are EGFP positive) x 4 x 10⁵ = 4 x 10³ positive cells.

4 x 10³ x 100 (dilution factor) = 4 x 10⁵ viral particles/ul

In general you should have at least 5 x 10⁵ viral particles/ul for embryo infections.