Mutagenesis of φX174 am3 cs70 incorporated into the genome of mouse L-cells

Abstract

The objective of our work with φX174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transfenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued ΦX174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of ΦX that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised ΦX DNA is recovered by column chromatography, ligated, and transfected into higly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10 −3. The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for ΦX am3 cs70, is close to one. Mouse L-cels containing the integrated ΦX174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 × 10 −5 (193 revertants in 1.4 × 10 7 phages). This is significantly higher than the 5.8 × 10 −7 reversion frequency of am3 (7 revertants in 1.2 × 10 −7 phages) among progeny phages rescued from untreated cells.