SRY GENE EXPRESSION IN PREIMPLANTATION MOUSE EMBRYOS

这个项目的目的是描述推定的 睾丸决定基因Sry在小鼠着床前胚胎中的表达及其意义 这一表达对于男性性别决定是否必要。这个 Sry mRNA的表达水平将在几个方面进行量化 基于聚合酶链式反应的植入前阶段研究 之前在我的实验室开发的程序(高德特和克雷恩， 1991年)。检测Sry在早期胚胎中的表达是否会导致男性性行为 检测反义核酸将被引入灭活 Sry mRNA，以及来自处理的活后代的性别 胚胎将在移植给寄主雌性后确定。这个 XY雌性的出现表明，在mRNA中的功能是 男性性别决定所必需的。几个“反义”策略将 被测试：(1)在含有化学物质的培养液中孵化胚胎 修饰和未修饰的寡核苷酸(ODN)；(2)微量注射 (3)注射反义RNA 从核探针载体中合成Sry mRNA的全长cDNA克隆 将从胚泡和睾丸中分离出核糖核酸及其序列 比较以确定是否使用差异剪接或启动子 在两种组织中产生不同的蛋白质。完整的 这些克隆的序列还将与基因组DNA进行比较，以 确定基因的结构并确定基因的位置 上游序列。我们的长期目标是研究Sry基因是如何 在早期胚胎发育中被激活，体内竞争分析将被 用于定位与该基因相关的顺式调控元件。

The aim of this project is to characterize the expression of the putative testis-determining gene, Sry, in preimplantation mouse embryos and to ask whether this expression is necessary for male sex determination. The level of expression of Sry mRNA will be quantified at several preimplantation stages using a polymerase chain reaction (PCR)-based procedure that was developed previously in my lab (Gaudette and Crain, 1991). To test whether Sry expression in early embryos causes male sex determination 'antisense' nucleic acids will be introduced to inactivate the Sry mRNA, and the sex of live offspring that derive from the treated embryos will be determined after transfer to host females. The appearance of XY females would indicate that functional in mRNA is required for male sex determination. Several "antisense" strategies will be tested: (1) incubation of embryos in media containing chemically modified and unmodified oligodeoxynucleotides (ODNs); (2) microinjection of modified and unmodified ODNs; (3) injection of antisense RNA synthesized from riboprobe vectors., Full length cDNA clones of Sry mRNA will be isolated from blastocyst and testis RNA, and their sequences compared to determine whether differential splicing or promotor use occurs to produce different proteins in the two tissues. The complete sequence of the cDNA clones will also be compared to the genomic DNA to determine the structure of the gene and to define the location of upstream sequences. With the long term goal of learning how the Sry gene is activated in early embryogenesis, in vivo competition assays will be used to locate the cis-regulatory elements associated with this gene.