Making Lentivirus using Invitrogen ViraPower T-TEX Lentiviral expression system

1. Clone gene of interest into pENTR/SD/D-TOPO vector

(1) Design primers:

Fw: CACC ATGG…….starting bp of your gene of interest

Rv: just reverse of your C terminal sequence, don’t forget to add stop codon

(2) Using pfx DNA polymerase ( or any other polymerase you used) to set up PCR reaction as other normal PCR, then use agarose gel electrophoresis to verify the quality and quantity of your PCR product.

(3) Purify PCR product using PCR purification kit from Qiagen

(4) Perform the TOPO cloning reaction: for optimal results, use a 0.5:1 to 2:1 molar ratio of PCR product: TOPO vector.

Reagent	Chemical Transformation
Fresh PCR Product Salt solution Sterile water TOPO vector	0.5-4 ul 1 ul To a final volume of 5 ul 1 ul
Total volume	6 ul

Mix gently and incubate for 5 minutes at room temperature

Place on ice and proceed to transform One Shot chemically competent E.Coli

(5) Transform One shot Chemically Competent E. Coli

a. Add 2 ul of the TOPO Cloning reaction into a vial of one Shot chemically competent E.Coli cells and mix gently

b. Incubate on ice for 5 to 30 min

c. Heat-shock the cells for 3o seconds at 42C without shaking. Immediately transfer the tube to ice.

d. Add 250 ul of room temperature S.O.C. Medium

e. Incubate at 37 C for 1 hour with shaking

f. Spread 50-200 ul of bacterial culture on a prewarmed selective plate and incubate overnight at 37C

(6) Analyzing Positive Clones

a. Pick 5 colonies and culture them overnight in LB or SOB medium containing 50 ug/ml kanamycin.

b. Miniprep DNA and analyze the plasmid by NotI and AscI ( make sure your gene of interest doesn’t contain these two enzyme restriction site)

(7) Send to sequence using T7 promoter primer and M13 reverse primer.

2. Getting of your gene of interest into to pLenti4/TO/V5-DEST vector:

(1) Setting up the LR Recombination Reaction:

(a)

Component	Sample	Positive Control
Entry clone ( 50-150 ng/reaction) pENTR-gus ( 50 ng/ul) pLenti4/TO/V5-DEST vector ( 150ng/ul) TE buffer, pH 8.0	1-7 ul -- 1 ul To 8 ul	-- 2 ul 1 ul 5 ul

(b) Remove the LR Clonase II enzyme mix from -20 C and thaw on ice.

(c) Vortex the LR Clonase II enzyme mix briefly twice , then add 2 ul into the reaction and mix well by pipetting up and down.

(d) Incubate at 25 C for 1 to 18 hours

(e) Add 1 ul of Proteinase K solution to each reaction, incubate for 10 min at 37 C.

(2) One Shot Stb13 transformation:

Just the same of normal transformation, the heat time is 45 seconds at 42 C

(3) Analyzing Positive Clones:

(a) Pick 5 colonies and culture them overnight in LB medium containing 100 ul/ml ampicillin

(b) Isolate plasmid DNA and analyze them by xba I and NotI ( make sure your gene of interest doesn’t contain them)

I usually didn’t sequence the plasmid, but if you want to, you can use CMV forward primer and V5 reverse primer

(4) Using Midiprep or Max prep to extract a large amount of DNA

3. Producing Lentivirus in 293 FT Cells:

(1) For each transfection sample, prepare DNA-Lipofectamine 2000 complexes as follows:

(a) In a sterile 5 ml tube, dilute 9 ug of the ViraPower Packaging Mix and 3 ug of the pLenti-based expression plasmid DNA ( 12 ug total) in 1.5 ml of Opti-MEM I Medium without serum. Mix gently.

(b) In a separate sterile 5 ml tube, mix Lipofectamine 2000 gently before use, then dilute 36 ul in 1.5 ml of Opti-MEM I Medium without serum. Mix gently and incubate for 5 min at room temperature.

(c)After 5 min incubation, combine the diluted DNA with the diluted Lipofectamine 2000 ( total volume= 3 mls). Mix gently.

(d) Incubate for 20 min at room temperature to allow the DNA-Lipofectamine 2000 complexes to form. The solution may appear cloudy, but this will not impede the transfection.

(2) While the DNA-Lipid complexes are forming, trypsinize the count the

293 FT cells. Resuspend the cells at a density of 1.2 X 10⁶ cells/ml in

Growth medium containing serum ( or Opti-MEM I Medium containing

Serum)

(3)Add the DNA-Lipid ( step 1d) to a 10 cm tissue culture plate with 5 ml of

Growth medium containing serum. Do not include antibiotics in the medium.

(4) Add 5 ml of the 293 FT cell suspension ( 6 X 10⁶ total cells) to the plate

Containing media and DNA Lipofectamine 2000 complexes and mix gently by rocking the plate back and forth. Incubate the cells overnight at 37 C incubator.

(5) The next day, remove the media containing the DNA-Lipofectamine 2000

Complexes and replace with complete culture medium containing sodium

Pyruvate( i.e. D-MEM containing 10 % FBS, 2 mM L-glutamine, 0.1 mM MEM Non-Essential Amino Acids, 1% penicillin/streptomycin, and 1 mM MEM Sodium Pyruvate).

(6) Harvest virus-containing supernatants 48 and 72 hours posttransfection by

Removing medium to a 15 ml sterile, capped conical tube.

(7) Centrifuge at 3000 rpm for 5 min at 4 C to pellet cell debris.

(8) You can concentrate the virus by centrifuge at 25,000 rpm for 1 hrs at 4 C to pellet the virus then resuspend it with a few ml of medium 50-100 times.

Then aliquote and keep in -80 C for long term use.

4. Transduction.

(1) The day before transduction ( day 1), trypsinize and count the cells, plating them in a 6-well plate such that they will be 30-50% confluent at the time of transduction, you can either culture them overnight or wait 4-8 hours to transduct the cells.

(2) On the day of transduction ( day 2), thaw your lentivirus stock and prepare 10 fold serial dilution to a final volume of 1 ml.

(3) Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells

(4) Add Polybrene (prepare a 6 mg/ml stock solution in deionized sterile water, filter , aliquote and keep at -20 C for long-term storage, maybe can store up to 1 year) to each well to a final concentration of 6 ug/ml, swirl the plate gently to mix. Incubate at 37 C overnight.

(5) The following day ( day 3), remove the media containing virus and replace with 2 ml of complete culture medium.

(6) The following day (day 4), treat cells as follows:

---For Zeocin selection( pLenti4/TO/V5-DEST or pLenti4/TO/V5-GW/lacZ stocks), remove the medium and wash the cells once with PBS. For each well of cells, trypsinzie the cells and replate the entire amount into one 10 ml plate containing complete culture medium with the appropriate amount of Zeocin to select for stably transduced cells.

---For Blasticidin selection ( pLenti6/TR), remove the medium and replace with complete culture medium containing the appropriate amount of Blasticidin to select for stably transduced cells.

(7) Replace medium with fresh medium containing antibiotic every 2-3 days

(8) After 10-12 days of selection ( day 14-16), you should see no live cells in the mock well and discrete antibiotic-resistant colonies in one or more of the dilution wells.

Note:

1. When performing the co-transduction procedure, you must transducer the Lenti6/TR lentiviral construct into mammalian cells before transducing the Lenti4/TO/V5-DEST expression construct to enable tetracycline-regulated expression of the gene of interest to occur. In addition, we generally wait at least 24 hours after transducing the Lneti6/TR construct before transducing the Lneti4/TO/V5-DEST constructed to allow time for the Tet repressor to be expressed.
2. Zeocin:

---Cell density can affect the efficiency of Zeocin selection. For the most efficient Zeocin selection, make sure that cells are not greater than 50% confluent.

----50-1000 ug/ml ( stock 100 mg/ml in water)

3. Blasticidin:

---stock 5-10 mg/ml in water

---Aqueous stock solution are stable for 1-2 wells at 4 C and 6-8 weeks at -20 C

--- Do not subject stock solution to freeze/thaw cycles

---Upon thawing, use what you need and store the thawed stock solution at 4 C for up to 2 weeks

---working concentration: 2-10 ug/ml