A SELECTABLE SYSTEM FOR MUTATION DETECTION IN THE BIG BLUE(R) LACI TRANSGENIC MOUSE SYSTEM - WHAT HAPPENS TO THE MUTATIONAL SPECTRA OVER TIME

Transgenic animals offer a powerful tool to study the mechanisms of sp ontaneous and induced mutagenesis in vivo. Herein we used a test versi on of a growth selectable assay to obtain spontaneous mutants in a lac I target transgene recovered from lacI transgenic B6C3F1 mice (Big Blu e(R)). This selection system may have certain advantages relative to t he more established plaque screening system for mutation detection bec ause: (1) the plating density of the phage is up to 60 times higher in the selectable assay, reducing the number of plates needed to be scre ened for a comparable amount of mutants; and (2) the mutant frequency obtained from the selectable assay is higher compared to the plaque as say, possibly due to a higher sensitivity for weaker mutants. However, the longer incubation time of the growth selectable assay might allow E. coli host derived mutants to appear. To address this issue, we inv estigated the sequence changes in the amino-terminal domain of the lac I gene of 405 mutants derived from the liver, spleen, brain, germ cell s and skin of five untreated 6-week-old mice. The mutant colonies were isolated after 60, 84, 108 and 150 h of incubation under growth selec table conditions. Tissue-specific differences in the mutational patter n obtained after 60 and 84 h disappear after a longer time of incubati on, possibly due to an increasing contribution of E. coli derived muta nts. The evolving selectable systems offer the potential to increase s creening efficiency, but the results suggest caution in interpreting d ata from this system because repair by E. coli of DNA lesions or misma tched heteroduplexes either originating in mouse in vivo or produced b y ex vivo manipulation as well as de novo mutations in E. coli might c ontribute significantly to the observed mutational spectra at each tim epoint.