We have recently released a substantially updated and improved version
of our protocols for generating gene knockouts in C. elegans. The
document can be downloaded from the "protocols" section of our Web site:
http://info.med.yale.edu/mbb/koelle/
Our methods are adapted from those originally developed at NemaPharm
and in the Plasterk, Barstead, and Moerman labs. Briefly, mutagenized
C. elegans are cultured in 96-well microtiter dishes. A mutant
"library" representing 920,000 mutagenized genomes is thus generated. A
portion of each culture is frozen alive and the remainder of each
culture is used to prepare genomic DNA. Any gene of interest will
suffer deletions of 100-1000 bp at a frequency of ~1/200,000 mutagenized
genomes. Using PCR, a genomic DNA sample containing such a deletion can
be detected, allowing the corresponding frozen microtiter culture to be
thawed. Thus live animals carrying a deletion mutation in the gene of
interest can be recovered.
Our methods require an initial investment of about 2 weeks of part-time
work to pilot the techniques followed by ~3 weeks of full-time work (if
two individuals work together) to construct a frozen mutant "library".
The library can be stored indefinitely and can be screened at least 200
times. Once the library is constructed one can isolate a live mutant in
a gene of interest in only 2-3 weeks of part-time work. Using these
methods we have so far succeeded in obtaining one to three mutant
alleles for almost every gene we have worked on.
The newly-released version of our protocols includes technical
improvements both in library construction and in methods for
PCR-detection of deletions. We have also included a new outline and
flowchart of the procedures, making them easier to understand. Our new
methods are substantially more successful than those we have previously
released. In the past we failed to isolate a deletion in about 1/3 of
genes we attempted to knock out. With the new methods we are virtually
always successful and can expect to obtain multiple alleles per gene.
We believe this technology is now successful enough that it is worth the
investment of time and effort for any small C. elegans laboratory.
--
Michael Koelle
Associate Professor
Department of Molecular Biophysics and Biochemistry
Yale University School of Medicine, SHM CE28A
PO Box 208024
New Haven, CT 06520-8024
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