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A Positive Selection Assay for Mutation Analysis in Big Blue,,,Animals

onditions (37C overnight) to determine the total number of rescued phage screened (figure 2, path B). Because the lLIZ shuttle vector contains the temperature-sensitive cI 857 mutation, both lcII + and lcII shuttle vector phages grow as plaques at 37C. Mutant frequency is determined as the number of lcII plaque-forming units (pfu) observed divided by the total pfu screened. The lcII selection scheme has been used to measure mutant frequency inductions in bladder tissue from mice treated with p-cresidine, with results comparable to lacI plaque color screening.4

The Big Blue Cyclist DNA sequencing kit can be used to rapidly sequence the lcII mutants identified with the l Select-cII assay. The cII target region is approximately 300 base pairs in length, including the cII mRNA ribosome binding site and the cII protein-activated PRE promoter.5,6 The relatively short length of the cII target region simplifies sequencing and expedites the analysis of mutational spectra.

Conclusions

The lSelect-cII mutation assay kit offers an alternative to the lacI plaque color-screening assay for mutation analysis in Big Blue transgenic rodents. Both assays feature unique advantages. The traditional plaque color-screening assay makes use of the larger, highly characterized lacI target region and benefits from the extensive database of experimental literature published over several years. By comparison, the lSelect-cII mutation assay is more cos
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