I have measured my physical titer of my lentiviral genome copies to be over 10^10, when taking an aliquot out of liquid N2. Despite this, when I treat fibroblasts with my lentiviral prep I have a transduction efficiency of around 1% or so. I use polybrene as suggested but that doesn't seem to help much. Could anyone offer any troubleshooting advice on lentiviral transduction for fibroblasts please?
I would advise you to recalculate the viral titer, if possible by cytometry. Title by PCR are usually much higher than the true title!
Ron Hrstka yes I have, the cells seem to get sicker but the protein of interest is rarely expressed, and even less so at higher doses.
Virginia Picanco thanks for your advice, good to know!
Hi Pavel, sometimes "less is more". I dilute the viral supernatant because too much virus is too toxic to cells so they don't transduce well. I do one infection with diluted viral sup (1:5 or 1:10) and I use 8 ug/mL polybrene. Next day I change the media and the other day, I start the antibiotics selection. I usually get close to 100% of transduction efficiency (basically no death during antibiotics selection). What fibroblasts do you use? I use this protocol with primary MEFs and NIH 3T3 fibroblasts. Hope it helps. Good luck!
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