INTRALABORATORY OPTIMIZATION AND STANDARDIZATION OF MUTANT SCREENING CONDITIONS USED FOR A LAMBDA LACI TRANSGENIC MOUSE MUTAGENESIS ASSAY .1./

A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has be en optimized and standardized for reproducible mutant detection. The m utagenic endpoints are blue lacI(-) phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques rem ain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant pl aque visibility. In vivo mutant frequencies for liver in untreated ani mals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10( -5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of intern al controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant fr equencies obtained when using standardized procedures.