lentivirus tranfection problem - (Oct/16/2007 )
We are trying to knockdown a gene in primary hepatocytes using lentivirus shRNA from Sigma. However, using the GFP-control, we are getting no transfection of hepatocytes using MOIs ranging from 1-100. The GFP-control seems ok as we see ok transfection of a standard cell line, although it is a lot lower than for our GFP-adenovirus. We have tried to increase the transfection efficiency using polybrene but this doesn't seem to help.
Does anyone have any ideas of how to get our primary hepatocytes to transfect using the lentivirus. Any comments you have would be great as we are new to this area.
what promoters are you using? Has the promoter known to work in hapatocytes?
another reason could be that the titres are low for the shRNA vectors. This is possible. Titre it on 293 cells or other cell line and compare it to gfp control.
Good Luck !!!
Try following which might help:
1. Try using Protamine Sulfate (final 8ug/ml) instead of polybrene.
2. Some primary cells get infected better when they are single cells in suspension and are spin infected.
3. Try double infection (Eg 48th hr and 72nd hr supernatant).
4. Quality of vector with gene of interset and packaging vectors is extremely important. Try a new batch.
maybe,the efficency will increase a little if you buy lipofectmine for it. best regard.