how the lentiviral vector transduct into cells? - ask a question (Feb/28/2007 )
Hi, dear all.
I bought several products of expression arrest-TRC human shRNA library. And I know they are lentivirus vectors inserted the shRNA I wanted.
The problems is how can I use them? Should I purify the plasmid DNA before I transfect them to my cells or I can directly use the vector to transduct to my cells without plasmid DNA purification?
Thank you very much!
Not sure I understand well...
You mean, you have shRNA already in a lentiviral vector? If so, you need to cotransfect your shRNA containing plasmid with two others plasmids coding for the envelope and the Gag-Pol viral proteins into permissive cell line (i.e. 293T). This will result in a virus production, with whitch you can infectec your cells and get the knockdown you want.
Otherwise, if the shRNA is a lentiviral plasmid and you want to only transfect this plasmid into a cell, I don't think it is possible.
Anyone else have an idea?
normally you need packaging cells for preparing the lentvirus which should be applied to your cells of study.
So the vector you probably hold will serve for transfection of packaging cells
You have to make virus from the lenti plasmids (with the help of packaging plasmids) or one could try transient transfection (use only lenti plasmid). Both would help. Virus prep might take time to optimise compared to transient transfection.
You need pure DNA for transfections.