Hi everyone,
I alreadydone co-transfection of Dharmacon Trans-lentiviral packaging mix and shRNA transfer vector into HEK 293T. The transfection efficiency was 90-95%. I proceeded with virus harvesting and virus concentrating. However, the viral titer that was achieved when i transduced the lentivirus into A549 lung cancer cell line was only 10^3-10^5 TU/ml. Thus, i used concentrated virus for transduction. I managed to get te highly expressed RFP for cell that was transduced with lentiviral-containing non-silencing plasmid. However, theRFP expression was really low for the cell that was transduced with lentiviral-containing shRNA vector. Can someone help me to troubleshoot this problem? And one more thing, is it possible for me to sort the RFP-expressing cell using facs and propagate them?
FACS sorting is a good start, but selection is necessary for getting integrated sequences in cells.
Have a look here to optimze your condition of transduction.
http://www.geg-tech.com/knowledge/protocols/transduction-with-lenti-one
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