A variety of methods such as injection, micromechanical piercing, and ballistic transofmation have been used to stably introduce transgenic DNA into C. elegans. While all the techniques have been successful for genes expressed in the soma, high-copy suppression and transgene-mediated co-suppression have made the expression of transgenes in germ line difficult. Of these methods the ballistic transformation rescue of
unc-119 has the potential to generate low-copy integrants, and even homologous recombinants, that express transgenes under endogenous promoters. While technically less demanding than injection the high material cost of maintaining large populations of C. elegans and the low frequency of animals with stable germ line expression, have been significant barriers. To resolve these issues, we have attempted to develop methods for generating large populations of
unc-119 animals and enrich for germ line expression by requiring it for viability or fertility.We have been successful in generating multiple integrated lines of C. elegans that express transgenes stably in the germ line using both the standard and modified versions of the ballistic transformation rescue of
unc-119 protocols. However, we found that the vast majority of lines rescued for
unc-119 did not have germ line expression of the transgene and when germ line expression was observed the majority of animals did not maintain transgene expression even if a potential integration event had occurred. Thus, the large number of lines rescued for the somatic
unc-119 marker but negative for germ line expression makes testing each positive line a cumbersome task. The expression of GFP fusion proteins facilitates the screening for stable germ line expressing lines, however, two issues make this approach problematic. First, lines with appropriate levels of GFP fusion protein expression below our detection limit will be lost. Second, constructs where GFP interferes with protein function cannot reliability used for downstream analysis. To reduce the number of false positives and eliminate the requirement of using a GFP fusion protein for screening we would like to develop a highly sensitive system for selecting only those animals with germ line expression. We reasoned that if germ line expression was required for fertility or viability the number of false positives should be greatly reduced. To test this hypothesis we have generated constructs carrying
unc-119(+) for primary selection and a secondary selectable marker either (
fem-3(+) or
emb-27(+)) that is required in the germ line. Our progress in the assessment of this dual selection system will be presented.