Platinum-A cell trasnfection toruble - (Oct/25/2011 )

Hi everyone,

So, this time I am trying to produce a retroviral vector containg hTERT gene, that will be further used for immortalization of primary cell lines.
I use Platinum-A cells
http://search.cosmob...02.20080327.pdf

I grew them in a 12 well plate and after they reached about 90% confluency I transfected them with the following plasmid: http://www.addgene.org/1773/ using Lipofectamine accroding to the manufacturer's protocol.
The following day I transfered the cells from each well (one well transfected with the plasmid and the other non-transfected) to a 25cm bottle. After 24h I added to both bottles hygromycine to the final concentration of 100ug/ml. After the first day the cells looked quite well in the transfected bottle and only few attached in the control. But on the second day(today) almost all the cells in both bottles are floating. However I kept them to see what will happen.

Actually a very similar thing happened to me previously but at that time the amount of plasmid added was less than specified in Lipofectamine protocol, and I laid the blame of my fail on this fact. But now I adjusted the amounts correctly, but still no result.

Do you have any ideas of which path I should take fot this problem?

Thank you in advance.

If I got you right, you are attempting to get a stable Platinum-A cells expressing hTERT, are you?
I am wondering if you ever treat those transfected cells with hygromycine prior to transferring them to 25cm bottle for scaling up? and do you observe the same problem when you do this?
Other wise, the cell dead you observed for the transfected cells might indicate that none of your cells have the gene stably integrated into the genome. They died because they have lost the plasmid. (PS: there must be at least a small population survive. Are you sure there are no viable cell after hygromycine treatment?)
Personally, I would prefer to select the stable clone in small tissue culture plate prior to scale up.