Worm Breeder's Gazette 12(4): 11 (October 1, 1992)
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.
In WBG 12, 2 (January 1992) we reported that we had started to set up a library of frozen cultures of the mutator strain MT3126 .The purpose of this library is to obtain Tc1 insertions in genes of known sequence; these can be analyzed to investigate gene function, or serve as starting points for introduction of subsequent alterations of the genome. The present size of the library is 3000 cultures; each culture is frozen in triplicate: twice in glycerol (for survival), and once as protK/SDS lysate (for DNA isolation). The number of survivors after thawing of a culture is 100-1000. Thus, the library now consists of more than 300,000 animals that can be recovered, a number high enough that we can expect Tc1 insertion alleles of most genes to be present.
The presence of Tc1 insertion mutants of a gene of interest in a culture can be investigated by a PCR, using one primer that is specific for Tc1 ,and one that is specific for the gene. As previously described we have pooled lysates in an ordered fashion and isolated DNA from the pools, so that a relatively small number of PCRs is sufficient to investigate the presence of a mutant in the whole library. We have changed the PCR method somewhat: previously we did one PCR, and enhanced the specificity by analyzing the products of the amplification reaction by Southern blotting, using the target gene as probe. We have now replaced the Southern blot by a second round of PCR with nested primers, after which the products can be analyzed on a regular agarose gel. Apart from the advantage of not having to work with blots, radioactive label etc., the important difference is that this method gives us DNA that can be cut out of the gel and directly sequenced. A small sequence track read from the Tc1 end is sufficient to determine the precise insertion site.
Thus far we have pulled out Tc1 insertion mutants in the following genes: gpa-1 , gpa-2 , gpb-1 , pgp-1 , pgp-3 ,and an anonymous gene from Jane Young and Ian Hope. Recently we have started to use primers that have been used for the sequencing of cosmid ZK637 (kindly provided by Molly Craxton and John Sulston) to investigate the presence of insertions in this area of the genome, and we have found insertions in ZK637 .7( lin-9 ),and ZK637 .2.Now that know how to do it, we can investigate the presence of a mutant in a day, and obtain the mutant animal and know the precise insertion site within a few days.
We do not want to make promises that we may not keep (such as knocking out all genes that are sequenced), but we can probably fish out Tc1 insertion mutants of pet genes of some other labs. We need a reasonable stretch of genomic DNA sequence, so that we can choose and synthesize nested primers that flank an area of coding sequences. We will have to find out in practice how many people we can help, and how many people would be interested.
We are working on strategies to use a Tc1 insertion as starting point for the isolation of e.g. flanking deletions. Quite a few of the insertions that we find are in introns (they are rich in the Tc1 target sequence TA), and these could serve for the isolation of null alleles.