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). 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 https://www.sigmaaldrich.com/sigma/product%...t/g1264pis.pdf), 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)