Quantifying in vivo somatic mutations using transgenic mouse model systems.
This chapter describes the use of the bacteriophage cII positive selection assay with the MutaMouse transgenic model system. The assay is similar to others involving a transgenic target, including the cII and lacI assays in the Big Blue(R) Mouse, lacZ in the MutaMouse, and the gpt delta assay. Briefly, high-molecular-weight DNA is purified from the tissue of interest and used as substrate during in vitro packaging reactions, in which the lambda transgenes are excised from the genome and assembled into viable phage. Phage containing the mutational targets are then adsorbed into an appropriate bacterial host, and mutations sustained in vivo are evidenced by either standard recombinant screening or selection assays. Mutant frequencies are reported as the ratio of mutant phage to total phage units analyzed. The lambda-based transgenic mouse assays are used to study and characterize in vivo mutagenesis, as well as for mutagenicity assessment. The models permit the enumeration of mutations sustained in virtually any tissue of the mouse and are sensitive and robust. Application of the assays is simple, not requiring resources beyond those commonly found in most academic laboratories.
Swiger
Roy R
RR
Midwest Research Institute, Palm Bay, FL, USA.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests; D009154:Mutation; D014157:Transcription Factors / Q000235:genetics; D014764:Viral Proteins
D014157:Transcription Factors; D014764:Viral Proteins; C039000:cII protein, bacteriophage lambda