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Stable transfection of SHSY5Y - (Jun/16/2008 )

Hi Guys!I'm new here and I need massive help. I've been trying to transfect SHY cells with a GFP tagged protein for the last 5mnths and create a stable cell line from there. My problem is 1) No matter the amount of Gentamicin I've added (highest amount now is 1mg/ml!!) the untransfected cells do not die off. The longest cultures I have was at 8weeks (in a 6-well plate) and although I had transfected cells proliferating the untransfected ones are not dead. These I had decided to passage because a) cells did not look healthy and b ) both transfected and untransfected cells were growing over each other. This passage was done roughly 2weeks ago and I still have alot of untransfected cells growing and only a handful of tranfected ones (much less from before I passaged them). unsure.gif Currently I'm doing selections with various amounts of Gentamicin (400ug/ml-1mg/ml). The other problem is the transfected cells do not seem to grow very much or at a considerable rate. Given the 8-week cultures only abt 1/5 or less of the dish area contained these cells.

Transfection reagent I'm using is PEI.Transfection media is as normal:DMEM/F12(1:1),Pen/Strep.L-Glutamax

Can anyone pls,pls help!!!I am very very desperate and have a supervisor that is getting frustrated as well!

Thanks in advance guys!!


eh, as far as I know, gentamycin is an antibiotic for prokaryotic cells, and not for eukaryotic cells (see also, so this makes it quite normal that the SY5Y cells don't die ... are you sure you're not mixing it up with geneticin (G418, a neomycin analog)?, cos this should work on SY5Y cells

which vector backbone are you using?

for the SY5Y cells, I have recently used the Fugene HD reagent with good results (although this is a lot more expensive than the PEI of course)