I am trying to overexpress a gene of my interest in EL4 ( T cell lymphoma) cell line using lentiviral transduction. I transfect 293T cells using lipofectamine 3000 and see weak GFP expression 48hr after transfection. I am concentrating the viral supernatant collected from 293T cells using PEG-it and use TransDux MAX from SBI system Biosciences for transduction of EL4 cells. I am consistently getting a few GFP+ cells (less than 2%). Moreover, GFP expression is not stable because sorted GFP+ cells are losing GFP expression even after culture in the high concentration of puromycin (10 ug/ml). I will appreciate if anyone can help troubleshoot this issue with EL4 transduction.
How many 293T cells are you using to produce virus? And how many cells are you infecting with your concentrated supernatant? You might just need to scale up your production and scale down your transduction.
Also PEG-containing concentration reagents are trouble. They cause cell membrane fusion and all sorts of stuff that will just wreck your target cells. If possible you should try to concentrate by ultracentrifugation.
Thanks for your response. I plate 1.5 million 293T cells per well of 6 well plate and use 3 wells (6 +6 ml media harvested at 24hr and 48hr after transfection=total 12 ml virus supernatant) to transduce 500,000 EL4 cells.
Not sure if using PEG-it is an issue, cells are healthy and happy after transduction, just donot have enough GFP+ cells.
Another issue is the resistance to puromycin. Please advise when should I start puromycin selection after transduction. I have tried different ways: the slow incremental increase in puromycin concentration 1>2>5>7>10 ug/ml puromycin but these cells eventually become resistant to puromycin. Please recommend what should be the starting puromycin conc and when to add it on cells after transduction.
1. I would use several 10cm dishes of 293T cells for production of lentivirus.
2. you would need to find a C. of puro at which just kill all the cells.
3. after transduction, 24-48 hours, trypsinized and reseed, add puro 24 hur post reseed.
I agree with the above. I wager you're not producing enough virus to be hitting 500,000 cells at an appreciable MOI. I think, at minimum, you should quadruple your scale of production and try hitting 100,000 cells with it.
I never used drug selection on EL4 cells. The lentiviral cassette was quite stable once integrated into this cell line. Maybe try transduction without the drug selection and see how your results are, especially if you have access to a sorter. I typically just re-sort my cells periodically to address any drift that might be occurring in my cells and that works beautifully.
Oh also you should collect at 48h and 72h post-transfection. Just do a media change at 18h post-transfection to get rid of transfection mix, but no need to collect at that time point.
We usually do transfections on 100 mm dishes.. Specially when the moi is too low. Transfection efficiency depends on the number of plasmids u use (1st gen vs 3rd gen). I once tried seq transfection to increase the moi. If you are using 3 plasmids system. Transfection one plasmid at a time keeping having transfection of envelop at the beginning. U can also make a stable cell using env with antibiotic pressure and then transfeft ur gene and hiv vectors. I would work more on increasing the moi. Alter the concentrations of plasmids. Use kits for purifying plasmids. Try spinoculation for transduction. After transduction do not add antibiotics concentrations towards the kill curve.. Do it gradually. Once the cells stabilize do a clonal selection. Take all the cells in 6 well and disperse them in 96 well such that each well has a minimum number of cells. Ideally one or two. Let them grow. Do not worry about media changes.. It will take a while for propagating. Find the well that has positive for expression.. Take all the cells and propagate.
- Badges
- Science method