My lab has had quite good success over the past several months knocking
out C. elegans genes. We use a procedure similar to that developed at
NemaPharm, in which mutagenized worms are grown in microtiter dishes,
stored frozen, and their DNA is screened for deletion mutations by PCR.
The method requires an initial investment of ~3 weeks of work to construct
the frozen mutant "library", which can be stored indefinitely and can be
screened about 200 times. Once the library is constructed one can isolate
a mutant in a gene of interest in only 2-3 weeks of work. We have so far
succeeded in obtaining one to three mutant alleles of every gene we have
attempted to knock out.
We think the method works well enough that many labs may want to try it.
We have prepared a very detailed protocol describing all aspects of the
procedure. We hope by making this available we can minimize the
difficulties other researchers might face when first trying this somewhat
involved technique. The protocol can be accessed via my lab's Web site:
<http://www.info.med.yale.edu/mbb/koelle/>http://www.info.med.yale.edu/mbb/koell
e/
--
Michael Koelle
Assistant Professor
Department of Molecular Biophysics and Biochemistry
Yale University School of Medicine
333 Cedar Street
PO BOx 208024
New Haven, CT 06520-8024
Tel: (203) 737-5808
FAX: (203) 785-6404
