I'm trying to get a stable knockdown of my target gene using 3rd generation shRNA lenti-vector within undifferentiated PC12 cells. The shRNA lenti-vector has eGFP and puromycin selection feature. PC12 is suspension cell but we coat cell culture dishes with collagen so those cells are attached to the dishes.
To knockdown my target gene, I directly transfected 2.5ug shRNA vectors into 6cm dish of PC12 (70% confluency) using lipofectamine 2000 and saw eGFP after 48hrs. The transfection efficiency is just 30% or less based on counting eGFP. Then after a few days 4ug/mL puromycin selection, most cells without transfection died, but I cannot find an effective way to pool out live cells. I tried to centrifuge with 800g x 10mins but a lot of died cells/debris came with live cells. Anyone can give some suggestions? Our lab does NOT have FACS.
Also, I tried to package lentivirus in 293T cell line and use those lentiviral particles to transduct PC12 cells but failed due to low efficiency. I can only see eGFP about 7-8days post transduction. If possible could anyone share some experience or send me a detailed protocol for transduction with PC12 please?
I appreciate any information!
According to Sigma, PC12 grows well in suspension, but attach to collagen (type IV, strictly - as you wrote). Do the selection using coated dishes. That is the simplest way to go through cloning in this case i think. Or you can use PLL/PDL (after transfection) if collagen is not stable enough.
Thanks Attila for your useful information.
Is PLL/PDL more stable than Collagen ? Do died cells come off or they still stick to the dish? If they still stick to dish then there is no way to collect live cells... and what concentration/how much should I use for coating the dishes?
Collagen shows some tendency to release from the base of TC dishes. Sometimes one uses two-times collagenization of TC dishes - if i remember well. For long term culture I suggest PLL for PC-12 like selection. My personal experience suggests that transfection works better on collagen than on a charged base material. So transfect a collagen-coated dish of PC-12 line then move the cells after a few days into a PLL-coated dish and start to select them by the antibiotic. That is very practical approach.
Died cells never attach. Only the live ones, if they have a proper surface.
PLL coating is easy, you can find some protocol on the net. I use PLO regularly, that is not the some, but similar, but i do not give the details just to avoid any problem.
Increase your transfection efficiency by succesful selection. PC-12 is not a well-transfectable cell line.
That is all. Good luck.
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