We are exploring methods for gene targeting. Such methods require the introduction of linear DNA fragments which can then be integrated into chromosomal DNA by means of homologous recombination. However, injection of linear fragments into the gonadal syncytium does not result in homologous recombination of the gene fragment and the chromosome. Rather, such DNA is assembled into extrachromosomal arrays. These arrays are most likely formed using the nonhomologous end joining pathway. We think that it is likely that the nonhomologous end joining happens in the cytoplasm, before the DNA has access to recombine with the chromosome. Thus, this pathway is in direct competition with the homologous recombination pathway that we hope to use in gene targeting. In order to achieve gene targeting we have been working to both increase the frequency of homologous recombination and decrease the frequency of nonhomologous end joining. In all cases, we injected a rescuing fragment from the
unc-18 gene into the
unc-18(
e234) strain and scored for rescued animals. DNA was injected directly into the nucleus of 5-10 oocytes per worm at a concentration of ~100ng/ul. The DNA could be incorporated in one of three ways: end joining into semi-stable arrays, integrating randomly into chromosomal DNA, or homologous recombination into the
unc-18 locus. Transformed animals were tested for integration by isolating true-breeding rescued lines. Homologous integrations were tested by outcrossing and testing for linkage.