retroviral cDNA libraries

Dr. Eugene S. Kandel ekandel at uic.edu
Tue Sep 12 11:37:31 EST 2000


Hi!

Clontech and Stratagene are selling retroviral libraries of
oligo(dT)-primed cDNAs for expression cloning. Both companies admit that
their inserts should include natural polyadenylation signals (usually
~20bp upstream of the actual polyA tail). If this is the case, a
substantial fraction of genomic RNA should be truncated in the middle,
leaving out the polypurine tract and  3' LTR. Consequently, such a virus
should be non-integratable, unless it undergoes a complicated (and
unlikely) rearrangement. If this happens, the library that is actually
delivered to the cells should be substantially enriched for rearranged
inserts, the inserts that were originally cloned in wrong orientation
(so polyA signal does not work) or the inserts that were internally
primed (polyA stretches in the middle of an RNA, etc.). If this
selective pressure is strong enough, it could increase the minimal scale
of the experiments to fish out rare full - length inserts or even make
the libraries useless altogether. 
Of cause, this may be a purely theoretical concern, but the tech
services of either company failed to provide any information indicating
that this is not happening. In fact, it appears, they have never done
extensive sequencing of the inserts recovered from the infected cells.
Has anyone successfully tried these libraries and sequence sufficient
number of inserts to estimate what fraction of those is still full-length?

Eugene.
-- 
Dr Eugene S. Kandel
The University of Illinois at Chicago






More information about the Methods mailing list