Worm Breeder's Gazette 13(3): 14 (June 1, 1994)

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.

Reverse genetics using transposon Tc1 :A progress report

Ronald H.A. Plasterk, Marianne de Vroomen

the Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121,1066 CX Amsterdam, the Netherlands, fax + 31 20 6172625, phone +31 20 5122081, E-mail RPLAS@NKI.NL

We have previously described a method for reverse genetic analysis of C. elegans, based on the isolation of a Tc1 insertion allele from a library of frozen worm cultures, and subsequent isolation of a derivative in which DNA has been deleted (Zwaal et al. (1991), PNAS 90, 7431-7435). Here are a few new points:

1. The current list of alleles isolated from our library is in the table on the next page. If you are interested in a mutant, we would suggest that you contact the researcher mentioned in the table. The data in the table (plus the precise insertion site sequence, and descriptions of possible phenotypes) are to be introduced into the ACeDB database.

2. We are still willing to screen the library for collaborators who send us primers for their gene of interest (see WBG 13, 26-27 for guidelines for primers). We handle requests in principle in order of arrival of the primers. The day we know that a line is obtained, we send an E-mail to the collaborator. On average it takes approximately 2 weeks full time work per mutant, but that time is spread over longer periods, since screens are done in parallel. There are three major sources of variation in the time required to find a mutant: 1. We initially screen with only one primer set, and look at insertions of Tc1 in one orientation; we do more screens when no address is observed. 2. Putative addresses are not always confirmed upon repetition. 3. The frozen cultures are oligoclonal, and the fraction of mutant worms in a "positive" culture varies from 50% to less than 1 %. In some cases the survival is low, and none of the survivors is found to have the desired mutation.

3. With a growing genome sequence, and a growing interest in reverse genetic analysis, we will not be able to keep up with the requests. A laboratory that foresees the need for many gene knock outs may consider to set up its own library. This is not so much work, the part of the library that we mostly use consists of only 3,000 frozen cultures. The new worm methods book, edited by Henry Epstein and Diana Shakes, will contain a chapter with detailed description of the method.

4. Alternative faster methods are not (yet) operational, we continue to work on those. One approach involves shotgun sequencing of Tc1 insertions in strains with high Tc1 copy numbers (R. Korswagen, M. Smit and RHAP, in progress). Anybody who has a strain containing many Tc1 elements, and who has reason to think that the insertions are independently acquired and therefore largely at new positions, is kindly requested to send us the strain and a description of its history.