Library rescue protocols
Ian A. York
york at mbcrr.dfci.harvard.edu
Thu Mar 2 15:48:10 EST 1995
We're planning to start trying to expression clone a mutant
gene. I.e. we have a mutant cell - COS-based - and we plan to transfect
in an expression library and select for reversion to wild-type. We have
no experience with this and I'm hoping I can get some advice.
Our first question is a general one. Who has
recommendations for or against particular libraries? -commercially
available, private, making our own, whatever: what is the best, what is
second best, what is right out?
Our problem is this. Our mutants show altered conformation
in MHC class I genes, but the structural genes are still there. (We are
looking at genes which impact on antigen processing - restoring the
mutant gene should restore the appropriate epitope.) We have preliminary
evidence that the effect is not the same with MHC proteins from different
species, and (getting to the point now) we are concerned that we will be
selecting for the trivial solution of expressing the human class I
structural genes. What we have decided to do, then, is first negatively
select - against human class I - then positively select - for
upregulation of monkey (COS) class I.
*Question* Anyone have any experience with negatively
selecting? Should we use panning (as for the positive selection) or
I'd also appreciate any general comments from people who have
worked with this. What are we likely to be missing? What step is going
to be the unexpectedly difficult one?
Replies either to this group or by E mail (york at mbcrr.harvard.edu)
If there are enough responses, I'll post a summary to the group.
Ian York (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328 Fax (617)-632-2627
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