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

冷冻精子库包括10,000只小鼠，总共携带30,000,000个ENU诱导的碱基替换。并构建了基因组DNA文库。这项合作研究的目的是建立一个系统，任何研究人员都可以通过这个系统从大规模冷冻精子库中自由和轻松地获得携带靶基因单碱基替换的小鼠品系。在4年的时间里，这个研究小组的成员为他们的目标基因设计了PCR引物，Gondo成功地发现了在他们的目标基因中携带ENU诱导突变的突变株。基于这个研究小组的进展，我们已经将该系统提供给日本国内外的任何研究人员。在为期4年的研究结束时，包括本研究成员在内的43名研究人员共申请了251个目的基因，数量是开始时的5倍。发现的突变数量已经超过了400个。通过体外受精的方法，Gondo复活了70多个突变系作为活鼠。因此，我们已经为用户提供了预期的突变株系，包括设计了PCR引物的所有研究成员。这证明，不仅制度建立得很好，而且我们取得的成就也远远超出了我们最初的预期。目前，我们和用户正在利用突变小鼠系进行基因功能分析。其中一些已经发表：例如，精神分裂症、抑郁症和多指畸形的模型鼠。总结已发现的突变谱。61%的编码序列突变为错义突变，10%为无义或剪接异常突变，29%为同义突变。考虑到1%的小鼠基因组是编码序列，冷冻精子库包括18万个错义突变和3万个无义或剪接异常突变。

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.