I want to do siRNA mediated inhibition of gene in COCs i.e. cumulus oocyte complex in bovines. I can't do the chemokine gene inhibition in cumulus cultures, as I have to further use the COCs for IVF. I read some reports in which they say that siRNA constructs using simple transfection agents is difficult for intact COCs. So they went for a adenovirus mediated shRNA method. But at present I have no facilities for a adenoviral mediated shRNA knock down. Can anyone suggest if I can get around >80 percent of transfection in cumulus oocyte complex intact cell. Also, in what time range we can observe the inhibitory effect of siRNA?
you can check this paper, the authors used the same protocol
http://www.ncbi.nlm.nih.gov/pubmed/24501174
Dear Asmita,
Are u targeting, before or after maturation?. I guess you are targeting before IVM. Their are several methods of transfection, but in your case lipid based method is may be suitable, you need to try lipo-siRNA gradient approach to optimized conditions to get best silencing. If you are making them denuded then is easy to do, but this is not your case. Also you need to think, how you get transfection in each and every cumulus cells of single oocyte, which is most critical issue. For analysis of silencing effect, you need to study in time line at different interval after completion of transfection. Have u been conduct any experiment.
Best wishes.
NLS
Respected sir,
Thank you so much for your kind response. I am targeting COCs before maturation and getting good efficiency is critical in my case, so i will have to check for the efficiency at different time intervals. Sir, i am going to start soon.
Thank you
Asmita
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude