Worm Breeder's Gazette 12(5): 21 (February 1, 1993)
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 this lab, we are analyzing cDNA clones of the worm systematically with respect to DNA sequence, gene location and expression pattern, aiming to identify all of genes of this organism and to understand the mechanisms of gene regulation in the life of the worm. To this end, we need an efficient way to disrupt gene functions of desired genes. Thus, we have been making a transposon Tc1 insertional mutant bank and have developed a strategy to get insertional mutants of desired genes, which are similar to those described by R. H. A. Plasterk et al. (WBG 12(4):11).
Construction of the mutant bank:
Pools of the mutant strain RW7097 ,each containing about 100 L1 s,were fed in 6 cm NGM-agarose plates to produce Fl progeny. One part of the Fl worms were made a duplicate of frozen stocks and genomic DNA were extracted from the another part. About 100 pools have been processed at one time in this way, producing processed 600 pools (60,000 independent worms) thus far. We are intended to make more than 1000 pools.
Isolation of Tc1 insertion mutants:
To test the quality of the bank, insertion mutants were searched for unc-22 and unc-54 genes. Aliquots of the DNA prepared from 12 individual pools were mixed and subjected to PCR analysis to detect the amplification specific to Tc1 insertion using one primer from Tc1 and another from a target gene (A. Rushforth ~ P. Anderson, WBG 11(5):65). Since a lot of non-specific PCR products were generated in the reaction, we have developed the following nested PCR strategy: For the first PCR, the target-gene specific primer was biotinylated at the 5'-end to purify the product amplified from the target region by using the streptavidinconjugated magnetic beads. The product bound to the beads were subjected to the second PCR using a set of nested primers and subjected to agarose gel electrophoresis. "Hot-start" of PCR by simply putting a reaction plate on the block of a PCR appratus pre-heated at 94°C also turned out to contribute to the reduction of non-specific products. For the mix of 12 pools which gave a positive band, the DNA from individual pools were analyzed as above to select a positive pool. From the frozen stock of the positive pool, about 200 worms were grown separately and the same PCR analysis as above was performed to finally identify mutant worms.
Thus far, we have isolated at least two mutants from the 200 pools for unc-22 and one mutant from the 600 pools for unc-54 .As to one mutant of unc-22 ,T c1 was inserted inside the fifth exon, and the typical unc-22 phenotype (twitcher) was found. We have confirmed co-segregation between Tc1 insertion and appearance of phenotype by crossing with N2 strain. In the latter case, however, the worms did not show any clear phenotype. As we have not yet mapped the Tc1 insertion point exactly, it may be inserted in an intron. Alternatively, as described by A. Rushforth & P. Anderson (WBG 12(4):14 ), splicing out of Tc1 region from mRNA which leads no appearance of phenotype might have occurred.