I have cloned my sgRNA in pLentiCRISPRv2 plasmid. I have tried lentiviral transduction of this plasmid into my ovarian cancer cell lines but the protein expression does not seem to alter. I am wondering maybe either the virus generation or subsequent transduction of cancer cells was not successful. Can anyone please provide me the detailed protocol? I would be very thankful.
Hi, I would check your lentivirus promoter. From the ovarian cancer lines I have used, some transduce very well and some barely at all. Also, the promoter heavily influenced transgene expression. From memory the SFFV promoter worked well
Jill Muhling
Hi, I have attached the map of my vector for the reference. Thankyou.
Wow, that plasmid is HUGE. From eyeballing it, it looks like the viral RNA transcript length is somewhere around 10.2 kb which is very large indeed. That is probably going to produce low titers.
It's not annotated but from the information on Addgene the promoter is EFS-NS. What's your ovarian cancer cell line?
I have seen previously that the SFFV promoter worked well in several ovarian cancer cell lines (including OVCAR3 and OVCAR5 if that helps). I did try the EF1a promoter in the OV90 cell line and it wasn't that great. I attached pics for you. That big plasmid combined with a promoter that's not very efficient in your target cells may be the reason why you're not having success.
- Badges
- Science topic