Hello
I'm producing lentivirus using HEK293FT cells.
The plasmid ratioI used was 4:3:1 with expression plasmid(pSIN backbone):p8.91:pMD.G.
I used calcium phosphate to transfect 293FT cells (10cm dish). I can see 70% cells are GFP positive after 24h. 48h and 72h later I collected the media (centrifugation/filtration .45uM), and put 1ml to my desired cell lines (N2a cells and 293FT) with 8ug/mL of polybrene. But I can't see any GFP expression 24h, 48h or even 72hr later.
I want to know what's wrong with my lentivirus production? I've tried it so many times.
Thanks!
I would sequencing the part that you introduce it to your cells to see weather genomic integration has happened or not by lentivirus.
good luck
Have you tried selecting your transduced cells? (e.g., puromycin selection) Transduction efficiency may be low but you can select for this population.
Also, how many N2a or 293 cells are you transducing with the 1 mL of virus/polybrene? Sometimes it's better to transduce fewer cells rather than many cells, that way you increase the likelihood that each cell in the population gets transduced.
- Badges
- Science topic