λselect cii mutation detection system

Big Blue^® mice or related lacZ mice (MutaMouse) have been successfully used to study both UVA & UVB-induced mutagenesis [60 (link), 61 (link)]. For mutation studies, WT and Pparg-/-^epi mice expressing the λLIZ transgene were anesthetized and treated with increasing doses of UVB. Control mice were not UVB treated. Five days later we euthanized the mice and excised the treated skin for epidermal DNA isolation. We waited 5 days before euthanasia to allow for both complete repair of CPD lesions and for clearance of mutations in non-cancer causing suprabasal cells through epidermal turnover (epidermal transit time = 3-5 days for hairless mouse epidermis) [62 (link), 63 (link)]. For DNA isolation, we separated the epidermis from the subcutaneous tissue using thermolysin as described above. Cells of the epidermis were isolated by trypsinization in 0.25% trypsin and the cellular nuclei DNA was isolated using the RecoverEase^™ DNA Isolation Kit (Agilent Technologies). Samples were dialyzed in TE buffer using G Biosciences Tube-O-Dialyzer. The mutation frequency was determined using Agilent Transpack Packaging Extract and λSelect-cII Mutation Detection System for Big Blue Rodents.