Genomic position influences spontaneous mutagenesis of an integrated retroviral vector containing the hprt cDNA as target for mutagenesis

We have used five isogenic human lymphoblastoid cell lines each containing a retroviral vector at a different position in the genome to assess the influence of these positions on spontaneous mutagenesis. The vector contains the hamster hprt cDNA and the neo gene, both genes are transcribed from the retroviral LTR promoter. The rates of mutation leading to a HPRT− phenotype during growth in non-selective medium differed up to 60-fold in the five retroviral integrates, ranging from 5.9×10−6 to 3.5×10−4 mutations per cell generation. From each of the cell lines approximately 20 independent mutants were analyzed by Southern blot analysis. In two cell lines all mutations were caused by inactivation of the LTR promoter (presumably by DNA methylation), whereas in another cell line the estimated rate of this mutation is 1000-fold lower. Another important class of mutation is homologous recombination between the LTRs. This accounts for at least half of the mutants in the other three cell lines. Mutants carrying deletions or point mutations form a minor fraction of the mutant distribution. Mutations confined to the hprt cDNA sequences only were studied by selecting HPRT− mutants in the presence of G418. Even for this subset of mutations the rates can vary at least 10-fold between the different genomic positions, ranging from 4.2×10−7 to 5.1 ×10−6. We conclude therefore that mutations leading to a HPRT− phenotype are quantitatively as well as qualitatively different in the studied cell lines. This suggests that spontaneous mutagenesis in a gene is dependent on its position in the genome.