A determination of whether restriction enzymes can induce specific deletions at the HPRT locus in CHO cells

Restriction enzymes are particularly informative clastogenic agents in that they produce only a single type of lesion, a double-strand break, at specific recognition sites in the DNA. Using a restriction map, particular restriction enzymes can be selected potentially to generate damage at a specific site in any target gene. We have used a variety of restriction enzymes to generate mutations in the hprt gene in Chinese hamster ovary (CHO) cells. The highest induced mutation frequencies have been with the restriction enzymes Sau3A I, which has a calculated cutting frequency in the genome of 1/338 base pairs, and Rsa I, which has a cutting frequency of 1/227 base pairs (Bishop et al., 1963). Both Sau3A I and Rsa i possess restriction sites in hamster hprt exon DNA, and it is likely that there are numerous recognition sequences in the large intervening introns of the gene which comprise nearly 97% of the hamster hprt locus. Mutants were isolated by selecting for resistance to the base analog 6-thioguanine. We have used the technique of multiplex PGR to amplify simultaneously all exons of the hamster hprt to screen induced mutants for specific deletions. Multiplex PGR consists of a series of oligonucleotide primers designed to amplify each of the exons of hprt using genomic DNA as template. Each amplified exon produces a discrete band when multiplex PGR products are resolved on an acrylamide gel. The absence of a single exon band or group of bands in a multiplex reaction is diagnostic of a deletion in hprt and also begins to define the size and location of the deletion. Multiplex PGR of restriction enzyme-induced HPRT-deficient mutants shows that both Sau3A I and Rsa I are capable of generating deletions in the hprt gene, but a different spectrum of type of deletions was induced by the two enzymes. RFLP analysis of exon specific bands and asymmetric PGR analysis was employed to determine whether the breakpoints in some of the mutant clones analyzed were at restriction sites. No changes were obsen/ed at restriction sites in hprt exons in the clones analyzed, and it was assumed that most breakpoints occured at restriction sites in the large intervening introns.