[Fluorescent-proteins] DsRed stable transfection doesn't Fluoresce?

Dr Mephesto via fluorpro%40net.bio.net (by dnhkng from googlemail.com)
Mon Nov 3 10:01:28 EST 2008


Hi,

I am have trouble with getting stable expression of DsRed in HEK
cells. I am using the <a href="http://www.ncbi.nlm.nih.gov/pubmed/
11730010">
pStoplight vector</a>  which express DsRed until it is cut out by Cre
Recombinase, and then GFP is expressed. I have cloned this into the <a
href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?
cmd=catProductDetail&entryPoint=adirect&productID=K601001&messageType=catProductDetail&showAddButton=true">Flip-
in kit</a> from Invitrogen to generate a stable cell line using flp
recombinase to integrate the vector.

It looks good for a while, but then, even though I have cells
surviving the selection agent, after a week or so, I dont have any
more red fluorescent cells!

So, I have no idea whats happening. It seems to me that either: 1) the
transient expression of flp recombinase is somehow acting on all the
stoplight transfected cells, somehow removing the gene... unlikely. 2)
DsRed expression is somehow being driven down by the cell? 3) DsRed is
somehow toxic and kills the stable cells, but then why does anything
survive!?

Anyway, any suggestions or advise would be very much appreciated!


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