Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts.
1998
Iriangle Park, NC 27709, USA Big Blue® Rat2 embryonic fibroblasts carry the λ-Liz shuttle vector which is also present in the Big Blue® mouse and rat. Mutations in the Big Blue® systems have most often been measured at the lacl locus. However, a method for positive selection of mutations at the λ cII locus was recently described. This assay appears to have many advantages over the use of lacI as a mutational target, but it has yet to be well characterized in mammalian mutagenesis studies. The objective of these studies was to determine the spontaneous and ethylnitrosourea (ENU)-induced mutant frequencies (MFs) and mutational spectra at cII using Big Blue® Rat2 embryonic fibroblasts. The average spontaneous MF was 13 ± 1.4 × 10 -5 . The average induced MF was 60 ± 10 × 10 -5 10 days following a 30 min treatment with 0.1 mg/ml ENU. Eighty four independent spontaneous mutants were sequenced: 23 (27.4%) were frameshift mutations and 61 (72.6%) were base substitutions. Two spontaneous frameshift hotspots were detected, both in mononucleotide runs. G:C→A:T transitions were the most common type of base substitution in cII; of these 71% occurred at CpG sites. The ENU-induced mutational spectrum at cII (44 mutants) consisted of 42 base substitutions (95.5%) and two -1 frameshift mutations (4.5%). Compared with the spontaneous spectrum, the ENU-induced spectrum had significantly fewer frameshift mutations (4.5 versus 27%) and base substitutions occurred predominantly at A:T base pairs (71 versus 34%). Overall, the spontaneous cII mutational spectrum reported here differs slightly from spontaneous spectra reported at the Big Blue® lacI locus, but the mutational spectra and base substitution MFs following treatment with ENU were comparable at both loci. These data support the continued use of cII as a selectable marker in mutagenesis studies involving cells or tissues that carry a λ transgene.
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