Library rescue protocols

[Ian A. York]

	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?

	Next:
	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 
biomagnetic beads?


	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.

	Thanks

	Ian
-- 
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