Detection and Characterization of Mutations in Mammalian Cells with the pSP189 Shuttle Vector System

Abstract

In the early 1980s the attraction of recombinant DNA technology was beginning to be felt in fields that had previously been refractory to molecular analysis. One such field was mammalian cell mutagenesis. Cloning technology offered the opportunity to recover mutant genes for which there were effective selection protocols (such as HPRT). Sequence determination of the entire gene could then display the nature of the mutations. Despite the feasibility of such protocols, the time and effort required discouraged most investigators. An alternative approach, based on shuttle vectors, appeared more attractive. These were plasmids whose design was based on advances in two fields. The extensive analysis of DNA tumor viruses such as SV40 had defined the genetic information necessary for viral replication in monkey and human cells. The biology and molecular biology of bacterial plasmids was also well developed. Thus, constructs with SV40 virus replication functions (the T-antigen gene and an origin of replication) linked to components of bacterial plasmids (the plasmid origin and a drug resistance marker) had been shown to replicate in monkey cells, and could be recovered and introduced into bacteria (Peden et al., 1980; Lusky and Botchan, 1981; these references also discuss the problem and resolution of the replication poison sequence found on pBR322). These experiments were primarily demonstrations of principle; there was no actual use of the shuttle technology. It seemed logical, however, to those interested in mammalian mutagenesis, to add a third component, a bacterial marker gene. The resultant vector, perhaps treated with a DNA damaging agent, would then be introduced into mammalian cells, allowed to replicate, recovered, and reintroduced into bacteria. Bacterial colonies with mutations in the marker gene would be recognized by standard microbiological selection or screening procedures, and the nature of the mutations identified by direct sequence analysis. This logic was quite compelling, and a number of groups set out to develop the technology.