TRANSGENIC MUTANTS AFFECTING SEX DETERMINATION AND FERT*

说明(申请人提供)：已知有一定比例的转基因插入会导致可识别的表型与实际使用的转基因无关。这是基因组重排的结果，通常是相对较小的缺失和/或倒位，伴随着转基因插入。这项应用旨在利用这一发现，并系统地识别和表征携带影响性别决定和/或生育能力的基因插入突变的小鼠。研究人员将使用一种新的转基因插入突变方案，通过在基因组中引入酪氨酸酶微型基因来挽救近交系FVB/N株的白化。根据他们从使用这种方法的小规模突变计划中获得的经验，10%-15%的插入会导致新的可识别的突变，其中10%-15%涉及生殖适合性，包括精子发生、卵子发生、性别决定和分娩。这种方法比使用化学诱变来鉴定繁殖突变体的表型驱动方案有许多优点。整个筛选过程，包括区分纯合子和杂合子，可以通过简单的肉眼检查毛色来实现。它自始至终使用单一的近亲繁殖品系，消除了任何令人困惑的遗传背景效应。与ENU突变不同，它不需要使用复杂的局部倒位或平衡染色体来帮助保持不育突变。最后，它最吸引人的特点之一是突变是由转基因标记的。这使得细胞遗传学图谱和快速的分子分析被破坏的座位。 考虑到这一新计划在产生生殖突变方面先前的生产性性质，以及在公共领域可获得小鼠基因组序列，研究人员建议以系统的方式检查更多数量的转基因小鼠。不仅将对繁殖突变体进行详细的鉴定和表型鉴定，还将获得它们的细胞遗传学图谱位置，产生侧翼序列，并确定突变在小鼠基因组中的确切位置。科林·毕晓普博士和保罗·奥弗贝克博士过去在分析表现出各种生殖表型的转基因插入突变体方面有相当丰富的经验。与男性生殖生物学领域公认的专家多洛雷斯·兰姆博士和女性生殖领域专家马丁·马祖克博士合作，研究人员认为，拟议中的计划将非常有成效。作为国家资源，该计划应该为生殖遗传学领域的研究人员提供一个独特的、高度详细的数据库，并获得新的小鼠品系。它也将与使用不同策略的其他诱变计划高度互补。

DESCRIPTION (provided by applicant): It is known that a percentage of transgenic insertions result in an identifiable phenotype unrelated to the actual transgene used. This is the result of a genomic rearrangement, usually a relatively small deletion and/or inversion, that accompanies the transgene insertion. This application is designed to exploit this finding and systematically identify and characterize mice carrying insertional mutations in genes affecting sex determination and/or fertility. The investigators will use a novel transgenic insertional mutagenesis scheme based on the rescue of albinism in the inbred FVB/N strain by the introduction a tyrosinase minigene into the genome. In their experience, gained from a small scale mutagenesis program using this method, 10-15% of insertions resulted in new identifiable mutations, of which 10-15% involve reproductive fitness, including spermatogenesis, oogenesis, sex determination, and parturition. This approach offers many advantages over phenotype-driven schemes employing chemical mutagenesis for the identification of reproductive mutants. The entire screening process, including distinguishing homozygotes from heterozygotes, can be achieved by simple visual inspection of coat color. It uses a single inbred strain throughout, eliminating any confounding genetic background effects. Unlike ENU mutagenesis, it does not require the use of complicated local inversions or balancer chromosomes to help maintain sterile mutants. Finally, one of its most attractive features is that the mutation is tagged by the transgene. This allows cytogenetic mapping and fast molecular analysis of the disrupted locus. Given the previous productive nature of this novel program with respect to producing reproductive mutants, and the availability of the mouse genome sequence in the public domain, the investigators propose to examine a greater number of transgenic mice in a systematic way. Not only will reproductive mutants be identified and phenotyped in detail, but their cytogenetic map position will be obtained, flanking sequences generated, and the exact position of the mutation in the mouse genome determined. Drs. Colin Bishop and Paul Overbeek have had considerable experience in the past in analyzing transgenic insertion mutants exhibiting a variety of reproductive phenotypes. Working together with Dr. Dolores Lamb, an acknowledged expert in the field of male reproductive biology, and Dr. Martin Matzuk, an expert in the field of female reproduction, the investigators feel that that the proposed program will be very productive. Designed as a national resource, this program should provide researchers in the field of reproductive genetics a unique and highly detailed database and access to novel strains of mice. It will also be highly complementary to other mutagenesis programs using different strategies.