Worm Breeder's Gazette 17(4): 16 (May 1, 2003)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Yale University School of Medicine, New Haven CT 06520
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.