RECEPTOR ISOLATION BY "PANNING"

Lindsay Collinson p8620253 at acsusun.acsu.unsw.edu.au
Wed Feb 21 23:57:08 EST 1996


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.

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

I am using the above vectors to construct cDNA libraries and to 
transform COS1 cells for use in expression cloning.

Analysis of plasmid DNA isolated following several rounds of
transfection, selection has revealed the presence of what appears to be
'deletion' plasmids which were confirmed to be of pcDNAI vector origin
by sequencing (appox. 2 to 3 kb in size compared to the full length
4.8kb vector). They contain the necessary genes for survival during
transformation into COS1 cells and antibiotic selection in E. coli.

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).

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

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

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

I welcome your opinions.

Lindsay Collinson





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