IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

Cloning receptor genes using the ligand?

Glen Gaughan gaughan at bms.com
Fri Nov 24 10:41:33 EST 1995

In article <49079m$rfb at news.cais.com>, rover at ns1.livnet.com (Rover) wrote:

>         We have heard in the wind  that there is a method somewhere for
> cloning receptor genes using the ligand or ligand information. If
> anyone has heard of this or knows of a reference please let me know.

There are many reports describing successful application of the expresion
cloning technique; most often the target is a peptide GPLR (endothelin,
angiotensin, interleukins) - have a look at the literature. The key
elements are (1) a good iodinated ligand,  (2) a good transient
transfection method, (3) a good library, (4) a good detection method, and
(5) patience. WRT the ligand it must have high specific activity and very
clean binding or it will be very difficult to determine true positives in
the 1st round of screening. (It should go without saying that it must have
high enough affinity/a slow enough off-rate so that you can bind, wash, &
dry w'out losing signal.) The need for an efficient transfection procedure
is obvious; it's critical to optimize for your particular batch of
reagents and cells. (3) is also obvious; (4) requires some comment. We've
found photoemulsion to be unnecessarily difficult both to setup and to
evaluate. We do use chamber slides for binding; however, rather than dip
the slides we just lay them down on film. Methods based on 'scraping and
counting' are not as sensitive as film. WRT (5) it really does pay to take
the time to carefully optimize your transfection method and binding
conditions. It also pays to take care in plating out the library; optimize
the transformation procedure (electroporate if possible), keep your pool
size reasonable, take some care with the plasmid preps from the pools.

More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net