Development of mutant mice carrying base-substitutions in target genes.

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Frozen sperm archive was constructed encompassing 10,000 mice carrying a total of 30,000,000 ENU-induced base substitutions. The genomic DNA archive was also constructed. The objective of this collaborative research is to establish a system by which any researchers may freely and easily obtain mouse strains that carry single-base substitutions in target genes from the large-scale frozen sperm archive. In the 4 years term, members of this research group designed the PCR primers for their target genes and Gondo successfully discovered mutant lines carrying ENU-induced mutations in their target genes. Based upon the progress in this research group, we have made the system available to any researchers within as well as outside of Japan. By the end of the 4 years term, 43 researchers including the members of this research requested a total of 251 target genes, the number of which is 5-fold more than that of the beginning. The number of discovered mutations had already exceeded 400. By using the in vitro fertilizing method, Gondo revived more than 70 mutant lines as live mice. Thus, we have provided expected mutant lines to the users including all the members of this research who designed the PCR primers. It vindicated that not only the system was well established but also we achieved much more than we originally expected. At present, we and users are conducting the analysis of gene functions by using the mutant mouse lines. Some of them have already been published : e.g., model mice for schizophrenia, depression and polydactyly. Summarizing the discovered mutation spectra. 61%, 10% and 29% of the mutations in coding sequences were missesnse, nonsense or splicing anomaly, and synonymous mutatons, respectively. Considering that 1% of mouse genome is coding sequences, the frozen sperm archive consists of 180,000 missense and 30,000 nonsense or splicing anomaly mutations.