Identify Sex Determination Genes By ENU Mutagenesis

描述（由申请人提供）：哺乳动物的性别决定是由一系列基因开关控制的，这些基因开关在发育的关键时期影响细胞命运和分化。值得注意的是，原始胎儿性腺是双电位的，生殖腺前体细胞可以沿着雄性（睾丸）或雌性（卵巢）途径发育，这取决于表达的基因。该项目的主要目标是确定调节性腺发育和表现性别的关键基因。为此，研究人员建议利用现有的西北大学全基因组ENU突变中心，筛选性别逆转小鼠，表现为XY表型的雌性或XX表型的雄性。该项目的具体目的是：目的1：筛选enu突变小鼠的后代进行性别逆转。小鼠的基因特征将取决于y染色体Sry缺失的存在与否。在21日龄时，它们将被分为雄性或雌性。这个屏幕每年将包括至少10,000只老鼠。目的2：研究性别逆转小鼠的性腺表型。形态学、激素、组织学和发育分析将用于描述与性逆转相关的性腺（睾丸和卵巢）缺陷。功能分析将评估男性的精子发生或女性的排卵。这些初步研究之后将使用原位杂交、定量RT-PCR、免疫组织学和western blot研究对改变的基因和蛋白质表达进行更详细的表征。微阵列分析将用于识别改变的遗传途径，特别是在性腺发育的关键阶段。目的3：延续与性逆转和性腺发育不良相关的突变的种系传播，并绘制缺陷的遗传位点。目标4：通过提供“在线”的表型筛选分析，作为国家资源的小鼠突变体。除了在网上列出已确定的表型外，假定的可遗传突变将被冷冻保存，并通过与已建立的国家分销中心的安排直接提供给科学界。

DESCRIPTION (provided by applicant): Sex determination in mammals is governed by a series of genetic switches that influence cell fate and differentiation during critical periods of development. Remarkably, the primordial fetal gonad is bipotential and the precursor gonadal cells can develop along either a male (testis) or female (ovary) pathway, depending on which genes are expressed. The main goal of this project is to identify key genes that regulate gonadal development and phenotypic sex. To this end, the investigators propose to screen mice for sex-reversal, manifest as XY phenotypic females or as XX phenotypic males, taking advantage of the existing Northwestern University Genome Wide ENU Mutagenesis Center. The specific aims of this project are: Aim 1: To screen progeny of ENU-mutagenized mice for sex reversal. Mice will be characterized genetically for the presence or absence of the Y-chromosomal Sry gone. At age 21 days, they will be classified as phenotypically male or female. This screen will include at least 10,000 mice per year. Aim 2: To characterize the gonadal phenotype of mice with sex-reversal. Morphologic, hormonal, histologic, and developmental analyses will be used to characterize the gonadal (testis and ovary) defects associated with sexreversal. Functional analyses will assess spermatogenesis in males or ovulation in females. These initial studies will be followed by more detailed characterizations of altered gene and protein expression using in situ hybridization, quantitative RT-PCR, immunohistology, and western blot studies. Microarray analyses will be used to identify altered genetic pathways, particularly during key stages of gonadal development. Aim 3: To perpetuate germline transmission of mutations associated with sex-reversal and gonadal dysgenesis and to map the genetic locus of the defect. Aim 4: To act as a national resource for mouse mutants by providing access to phenotypic screening analyses "online". In addition to listing identified phenotypes online, putative mutants that are heritable will be cryopreserved and made available to the scientific community directly from the Center and through arrangements with an established national distribution center. Gonadal development provides an excellent opportunity to identify genes involved in differential organogenesis. In addition to providing new information about basic mechanisms that regulate gonad development, these studies are also likely to enhance our understanding of gonadal dysgenesis and infertility in humans.