RECEPTOR ISOLATION BY "PANNING"

Richard Aaron Warnock erawtech at leland.Stanford.EDU
Thu Feb 22 16:56:50 EST 1996


In article <4ggt34$761 at kettle.magna.com.au>,
Lindsay Collinson  <p8620253 at acsusun.acsu.unsw.edu.au> frustratedly wrote:
>
>Is there any one out there who has had experience with the Invitrogen
>expression vectors pcDNAI or pcDNAI/AMP and/or experience with
>expression cloning/panning using COS1 cells.  We are using the 'panning' 
>technique of isolating a receptor for a novel cytokine.

Yep. I suspect there are thousands of us... 

>We are having problems with the stability of our plasmid library...

You aren't the first, nor shall be the last...

>Analysis of plasmid DNA isolated following several rounds of
>transfection, selection has revealed the presence of what appears to be
>'deletion' plasmids

We all 'feel your pain'...

>I have used two libraries [one in pcDNAI (prepared by invitrogen) and
>the other in pcDNAI/AMP (prepared in-house] and both are producing
>similar deletion clones (the pcDNAI library in particular).

Sounds all too familiar...

>I have a feeling that repeated transformation of selected clones into
>COS1 cells induces some sort of plasmid recombination/instability
>resulting in plasmid deletion.

Please consider your feeling fact!

>We transform COS1 cells using the Life Technologies transfection reagent
>'Transfectamine' and cycle clones into E. coli DH10B cells (also from
>Life Technologies.

Doesn't matter how you do it (x-fect, that is), it's endemic to this
technique, especially in these cells...

>Has anyone experienced similar problems with these vectors or has had
>experience with expression cloning using COS1 cells?

Yes! Dare I say this problem is well known? Perhaps just not commented
on enuff...

>I welcome your opinions.

Aaron's top 7 suggestions:

1. Give up science (ok, maybe just mol. bio.).

2a. So you decide to stay at the bench; what a glutton! Try to limit
your rounds of selection and retransfection (eg pools...)

2b. Limit time between xfection and selection (you can check how fast
stuff comes up with a control gene you've an antibody too. In fact,
we've done tons of analysis with controls to see when and where we
lose things. Make sure you can pull out a known...)

2c. Vary the chloroquine, if you use it.

2d. Try the old spheroplast fusion on the 3rd round. This severely
limits the number of *different* plasmids in each cos cell, apparently
limiting the ability of the small rapidly reproducing plasmids from
out-producing the others...)

3b. Clone your cos cells by limited dilution, and find a subline with
better stability... (Yep, a pain, but it works. Wish I had something
to send you, but I'm out of the biz...)

3b. Next try different Cos parent lines, or totally different cell
lines (that can support episomal replication, of course).

4a. Try clever selection techniques. (eg plasmid or insert size, etc)

4b. (This is a stab in the dark for me, since I don't know what type
of "novel"ty you have...) Use multiple selection during initial
panning. Now, if you were using an monoclonal, you could start with
poly or a mixture of monoclonals. Since you're using a peptide (I
think), consider exploiting any characteristics of related cytokines
(if any).

5. Consider the fact that many receptors are multimeric. Make sure
you've decent affinity with your peptide or whatever to get your
target, by affinity column, blotting, whatever (assuming you know the
target cell/tissue - eg where-ever you made your library
from). Hopefully you've a guess about the receptor structure. If you
can at all isolate any material, make antibodies and go back to
suggestion 4b.

6. Curse your luck, try again, and/or call Brian S. and complain
bitterly. Maybe he'll say, "Send me your peptide, and I'll send you a
clone!" Yeah, right.

7. Wear the correct expression cloning mojo. (I prefer the shredded
bits from an old Maniatis in a well worn leather tobacco pouch!)

All the best-

Aaron ("The Cynical Boy") Warnock

(who's now a cell biologist...and cautions anyone from taking
science too seriously, or his advice as gospel!)

-- 
"Nothing more is needed to destroy a man, than the conviction that his
life's work is useless."  -Antonin Artaud

erawtech at leland.stanford.edu (R. Aaron Warnock)



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