Relative sensitivity of the endogenous hprt gene and lacl transgene in ENU‐treated big blue™ B6C3F1 mice

Abstract

Three-week-old Big Blue (BB) B6C3F1 mice were given a single i.p. injection of ENU. Three weeks later, splenic T cells were isolated from each animal by ficoll gradient centrifugation and divided into two samples. One sample was cultured to measure hprt- mutation and the other was used to extract DNA for lacI- analysis. T cells from BB mice exposed to 0, 4.5, 13.5, and 40 mg ENU/kg (9 or 10 animals per group) displayed dose-related increases in the frequency of both hprt- and lacI- mutations. Within each treatment group, the ENU-induced mutation frequency (average observed mutation frequency minus average control frequency) was remarkably similar at the two loci. This suggests that treatments that increase mutation frequency at the endogenous hprt gene also produce similar incremental increases at the BB lacI transgene. However, because of the ten-fold higher spontaneous mutation rate at lacI, the fold-increase over background produced by ENU at this locus was significantly less than the fold-increase produced at hprt. For example, the 4.5 mg ENU/kg treatment produced a 5.2-fold increase above background at hprt (P = 0.001), whereas only a 1.5-fold increase was produced at lacI (P = 0.140). Consequently, mutagenic insults that produce up to a fivefold increase in mutation frequency at an endogenous locus may be difficult to detect at the lacI transgene. Finally, the ENU-induced response at hprt in BB mice was identical to that in generic B6C3F1 mice, suggesting that there are no inherent differences between transgenic and normal mice in their response to this mutagenic agent.

Epigenetic regulation of genetic integrity is reprogrammed during cloning

“…It remains unexpressed and hypermethylated (18) in all tissues examined, and this facilitates a uniform analysis of the relative frequency of mutational events in different tissues or in different individuals subjected to different circumstances (e.g., reproduction by natural conception or cloning). In addition, spectra and frequencies of mutations in the lacI transgene have been directly compared to those in endogenous mouse genes including hprt and Dlb1, and were found to be similar for all types of mutations other than large deletions or insertions, which are not well detected by the lacI transgene system due to packaging constraints of the lambda system, and were not the focus of our analysis (31)(32)(33). Indeed, the extensive previous use of this system to determine the frequency and spectrum of point mutations in a wide variety of circumstances has rendered this the single best characterized target gene with regard to mutagenesis (24).…”

Mutation frequency declines during spermatogenesis in young mice but increases in old mice

“…It has been reported that the lacI transgene has a high spontaneous incidence of mutagenesis (19). Because the majority of spontaneous lacI mutations have been identified as GC 3 AT transition mutations that occur predominantly at CpG sequences, it has been hypothesized that these particular mutations may result from the deamination of 5-methylcytosine residues in CpG dinucleotides (1).…”

Exalnination of lacZ Mutant Induction in the Liver and Testis of Muta Mouse following Injection of Halogenated Aliphatic Hydrocarbons Classified as Human Carcinogens.

“…We have experienced that the probability was not low enough 15,28) to incorporate into the experiment of animals containing proliferated progeny cells of pre-existing mutation which had been generated during development before or after fertilization. The efficiency of repair on nonexpressed genes is relatively low 29) , and spontaneous mutation frequency is higher in transgenes than in endogenous locus 30) . Mouse numbered 607 was very likely to be a germ cell mutant, but sequence determination is required to confirm the clonal expansion of somatic mutation in the animal coded 626.…”