A plasmid rescue to investigate mutagenesis in transgenic D. melanogaster

Abstract

We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo. Transgenic Drosophila lines were established by tranformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis. The target gene cen be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation on the recircularized shuttle vectors. The resulting circular plasmids are then transformed back into E. coli lacZ⁻ mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal. The number of inactivated versus intact lacZ genes directly indicates the mutation frequency. By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely. Spontaneous background mutation rates using this system are 2.6±0.6×10⁻⁴. Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8±0.6×10⁻⁴ for 0.5 mM and 6.9±1.2×10⁻⁴ for 1 mM ENU, respectively.