Identify Sex Determination Genes By ENU Mutagenesis

描述(申请人提供)：哺乳动物的性别决定由一系列影响细胞命运和发育关键时期分化的基因开关控制。值得注意的是，原始胎儿性腺是双潜能的，前体性腺细胞可以沿着男性(睾丸)或女性(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型性别的关键基因。为此，研究人员建议利用现有的西北大学基因组宽ENU突变中心，筛查性反转小鼠，表现为XY表型雌性或XX表型雄性。本项目的具体目标是：目标1：筛选ENU致突变小鼠的子代进行性别逆转。小鼠将被定性为Y染色体Sry是否消失的遗传学特征。在出生21天时，它们将被归类为表型雄性或雌性。这一屏幕每年将包括至少10,000只老鼠。目的2：研究性反转小鼠的性腺表型。将使用形态、激素、组织学和发育分析来表征与性反转相关的性腺(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。在这些初步研究之后，将使用原位杂交、定量RT-PCR、免疫组织学和蛋白质印迹研究对改变的基因和蛋白表达进行更详细的表征。微阵列分析将被用来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：维持与性反转和性腺发育不全相关的突变的种系传播，并定位该缺陷的遗传位点。目标4：通过“在线”提供表型筛选分析，为小鼠突变提供一个全国性的资源。除了在网上列出已确定的表型外，还将对可遗传的假定突变进行冷冻保存，并通过与已建立的国家分配中心的安排，直接从该中心向科学界提供。 性腺发育为鉴定差异器官发生相关基因提供了极好的机会。除了提供有关调节性腺发育的基本机制的新信息外，这些研究还可能增强我们对人类性腺发育不全和不育的理解。

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.