Dear Vijay Barathi Elango!

Here you has the method!

Methods

Cell cultures

Cell lines used in this study were a fetal rat liver carcinoma cell line, FRL 19; a human embryonic kidney cell line, 293 and its derivative, 293T; and a murine embryonic fibroblast cell line, NIH 3T3. FRL 19 was maintained at 37°C in Ham and Dulbecco's modified Eagle's medium (1:1 ratio, DMEM; Life Technologies Inc) containing 2 mM glutamine, 4% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 μg of fungizone per ml (Ham and DMEM), 10-7 M of insulin, and 10-7 M of dexamethasone in a 5% CO2 incubator. All other cells were maintained at 37°C in DMEM containing 2 mM glutamine, 10% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, similarly in a 5% CO2 incubator. 293, 293T and NIH3T3 were maintained in DMEM containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomcycin at 37°C similarly in a 5% CO2 incubator. Cells were seeded at 5 × 105 on 10 cm or 7.5 × 105 on 15 cm plate and were at 70 – 80% confluence at the time of transfection or transduction.

Viral vector production

Replication-defective retroviral particles were generated by transient co-transfection of 293T cells with the three plasmids (pHR' CMVGFP or pHIV-CSGFP, pCMVΔR8.2 pr pCMVΔR8.9 and pHCMV-G), using a CaPO4 precipitation method as we previously reported [21]. Briefly, 293T cells were grown on 10 cm plates to 70–80% confluence and co-transfected with 10 μg pHCMV-G, 10 μg pHR' CMVGFP or pHIV-CSGFP and 20 μg pCMVΔR8.2 or pCMVΔR8.9. The plasmid DNA was diluted into 250 mM CaCl2 in 1/10-TE buffer (1 mM Tris HCl, 0.1 mM EDTA, pH 7.6) in 0.5 ml before an equal volume of 2× HBS (140 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, pH 7.05) was added and mixed by gently bubbling air through the mixture for 1 min. The solution was then added drop-wise onto the cells (100 μl per 1 ml of culture media). The cell cultures were rinsed with PBS and given fresh media within 10–12 hr after initiating transfection. The medium was harvested 48 hr post-transfection, centrifuged at low speed to remove cell debris and filtered through a 0.45 μm filter. The supernatant was stored at 4°C no more than 24 hr before it was used for transduction.

Ultracentrifugation

This was performed as reported previously [20,21]. Briefly, 30 mL of vector-producing cell (VPC) supernatant was added to each polypropylene ultra-centrifugation tube (6 × 30 mL), and ultracentrifuged at 50,000 g for 2 hr at 4°C on AH629 rotors in a Beckman refrigerated centrifuge. After centrifugation, the tubes were promptly removed and supernatant decanted. The viral pellet was resuspended in 0.6 mL of DMEM and stored at -20°C.

In vitro transduction and determination of lentivector titre

This was performed as we previously reported . Briefly, cultured 293T cells were seeded at 5 × 105cells and transduced with serially diluted and concentrated viral vector stocks 16–18 hours after seeding when cells were about 70% confluent. For each transduction, 8 μg/mL of polybrene (Sigma) was included in the transducing inoculum. Forty-eight hours after transduction, EGFP positive fluorescent cells were counted using epifluorescent microscope (Nikon eclipse E600, Japan) with the fluorescein isothiocyanate (FITC) excitation-emission filter set at 470 nm. The viral vector titre was determined as the average number of EGFP positive cells per 20 1-mm2 fields multiplied by a factor to account for dilution of the viral stock as well as plate size and thus total cell number. Alternatively, 48 hours after transduction, cells were harvested, resuspended and sent for FACs analyse at a local FACS facility (Queensland Institute of Medical Research, QIMR, Brisbane, Australia).

Transduction – studies of target cell volume and number

293T cells at 1 × 103, 3 × 104 or 1 × 105 per well were seeded in triplicate in 24-well plates. Transduction was performed with the same stock of viral supernatant using volumes of 100 μl, 300 μl and 1 ml for 2 hours in the presence of 10 μg/ml polybrene. After the incubation period the cells were washed with fresh growth medium twice and allowed to grow for 2 days before the cells were trypsinised and fixed with 2% formaldehyde + 0.2% glutaraldehyde in PBS. EGFP positive and total cell numbers were counted with a haemocytometer using epi-fluorescence microscopy.

Transduction – studies of variable vector-cell contact and adsorption periods

293T cells were grown in 24-well plates to approximately 70% confluence. The cells were incubated with 500 μl of pHR' CMVLacZ supernatant for 10 min, 30 min, 1 hr, 2 hr, 4 hr and 17 hours. After the indicated incubation period the viral supernatant was removed and replaced with fresh media. Forty-eight hours post-transduction, cells were stained to check for the presence of LacZ with the following solution: 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6.3H2O, 2 mM MgSO4, and 1 mg/ml X-gal in PBS. Blue cells or colonies were counted as positive for gene transfer.

Transduction – studies of vector decay

Cell-free viral vector-containing supernatant was incubated at 37°C for 30 min, 2 hr, 4 hr, or 24 hr prior to being used as the transducing medium (500 μl), with experimental samples in triplicate. 293T cell at 70% confluent cultures were exposed to the transducing media for 2 hours, after which the inoculum was removed and the cultures replenished with fresh media. Forty-eight hr post-transduction, cells were stained to check for the presence of LacZ with the X-gal solution. Blue cells/colonies were counted in 3 fields and the average used as the titre at that time point.

Transduction – studies of MOI and transgene expression

293T cells were plated in a 10 cm plate at 1 × 105 cells/plate. Transduction was performed with viral vector stocks at a MOI of 2, 4, 8, 16 and 32 in the presence of 10 μg/ml polybrene (Sigma). Transduced cells were passaged every three days and EGFP positive cells sorted at a local FACS facility (QIMR, Brisbane, Australia).

Flow cytometry

Flow cytometry analysis was performed to evaluate the expression of lentivirus vector-mediated gene transfer. Cells were washed with PBS, and then fixed with 1% paraformaldehyde before the analysis. Samples were analysed on a FACScan flow cytometry in QIMR.

I hope, that the answer is usefull for you!

Sincerely yours!

Dr. Bratko Filipič

E-mail: [email protected]

In my case it works better using RT PCR than FM. Can you try RT PCR for your titre estimation.

Dear Dr.Filipic Bratko

Thank you very much for a detailed reply. I did get some useful information regarding viral supernatant handling and transduction from the method you shared.

I noticed that for transfection of HEK cells, CaCl2 method was used. I have used Lipofectamine, not sure if that affects (toxicity) the packaging of retrovirus. And, I dont go for ultracentrifugation of the supernatant. I just mix the 48hr and 72 hr supernatant (16ml) with a retrovirus precipitation solution (4 ml) and keep in overnight at 4'C and spin them down the next day only at ~1500 g ( too low ?) this is the protocol suggested by the manufacterer (Alstem, Retrovirus precipitation solution )

Also, I find that the concentration of Polybrene is 8 ug/ml or 10ug/ml , while in my case i used 4ug/ml. Nice information about vector decay and vector -variable contact, I think the problem in my protocol most likely exists at the concentration of supernatant using the precipitation solution. I guess I should eventually consider ultracentrifugation.

I know this is an old topic, but I'm curious if you eventually found out what the problem was and how you solved it.

Especially, because I can think of other reasons that could result in no transgene expression.

Hi Rajiv, there wasn't a major technical issue with the plasmid as such (kindly share technical speculations). Eventually it had to do with polybrene being added to the media ( in addition to viral Supt resuspension media). And it worked.

Thanks