What are the prospects that you can advise me about my intent to
immortalize primary cultures of human urothelial cells with the
retroviral construct (pLPC-hTERT)?
I received this construct from Geron via a MTA. My lab at Children's
Hospital is very good at growing all kinds of uro cells: renal pelvis,
ureter, and bladder. But because they are primary cultures, they only
useful for about 40 doublings (5-6 passages). While a constant supply of
tissue isn't a problem, I'd like to be able to guard against that
scenario -- hence my interest in immortalization.
In talking with the techs at BD Biosciences (Clontech), they are
recommending a packaging cell line called RetroPak PT67 because of my
desire to generate a stable line that I will characterize (titer, etc)
and freeze down in liquid N2. I foresee a need for titered virus over
the next few years and would like to jump through the packaging hoops
just once.
My concern is that this line will give about 1x10^6 cfu/ml. This
might be marginal for my urothelial cells. Do you agree? Is there a way
to concentrate the conditioned medium prior (to increase virus
concentration) prior to infection of the primary uro cells?
There is a Pantropic packaging line that they sell (10^8), but it
isn't licensed for human cells. This is too bad. Are there other lines
that will give me both stability AND highest titers?
The pLPC-hTERT construct will require puromycin to select against
non-infected cells.
Thanks in advance,
Jim Bassuk, PhD
Univ Washington - Urology
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