DEVELOPMENT OF A RAT-CELL LINE CONTAINING STABLY INTEGRATED COPIES OFA LAMBDA-LACI SHUTTLE VECTOR

A rat embryo cultured cell line was generated that carries stably inte grated copies of a lambda/lacI shuttle vector, containing the lad gene as a mutational target. After the desired treatment of the cells, thi s vector can be rapidly and efficiently recovered from the cell DNA by in vitro packaging and then screened for mutations in the lad gene, u sing bacterial detection systems. The vector is identical to that inte grated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis. Characterization of the cell line by fluoresce nce in situ hybridization showed that the phage DNA is integrated at t wo distinct sites on separate chromosomes at approximately 50-70 copie s per cell and the cell line is polyploid. The rescue efficiency is ap proximately 100 000 pfu/mu g of genomic DNA. To examine the ability of the cell line to detect mutations in the lad gene, the cells were tre ated with 100 mu g/ml of the direct-acting alkylating agent N-methyl-N -nitrosourea (MNU) for 30 min at 37 degrees C and grown to confluence. The shuttle vector was rescued from untreated and mutagen treated cel ls, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10(-5) and 92.7 x 10(-5), respectively. The cell line can be used to detect mutations in the lad gene, followed by recovery of muta nts for sequence analysis. The cell line may be valuable for short-ter m in vitro mutagenesis studies, oncogene and tumor suppressor studies, and DNA repair studies.