Screening for genes essential for development of the mouse embryo

我们正在使用两种方法来鉴定和分离参与哺乳动物胚胎和胎儿的调节生长和分化的基因。其中第一种是插入性突变，通过将外源DNA插入到基因中产生突变表型。这也在突变位点创建了一个分子标签，提供了对基因的直接访问。几年来，我们分析了来自胚胎干细胞（ES）的转基因小鼠品系，这些胚胎干细胞在组织培养中被逆转录病毒感染以产生插入突变。我们筛选了40多个前病毒插入，专门针对导致产前致死的突变，表明该基因对正常发育绝对必要。该筛选确定了4个隐性产前致死突变，与前病毒插入密切相关，并可能由前病毒插入引起。目前我们的研究重点是由412-r原病毒引起的插入突变。我们分离出了cosmids中的412-r逆转录病毒插入位点和周围的DNA。利用外显子捕获获得候选基因片段，并通过5‘和3’ RACE延长，用于探测胚胎cDNA文库。这些努力使我们能够确定突变基因的候选基因。该原病毒位于Sumo/sentrin特异性蛋白酶1 （SENP1）基因的第一个内含子中，SENP1是最近发现的一种参与Sumo酰化调节的蛋白质。Sumo/sentrin是一种与泛素相关的小蛋白质，它以类似泛素化的过程被添加到蛋白质中。SENP1的作用是将Sumo从修饰蛋白中去除。我们关于412-r突变SENP1基因的证据来自纯合子的表达水平分析，其表达水平降低了约50%。SENP1通常是普遍表达的。病毒插入导致所有细胞的表达水平降低，或者仅在某些细胞或组织类型中完全下调该基因的表达。目前，我们正在通过突变胚胎的原位杂交来评估这一点。我们对412-r突变表型的持续分析将提供洞察SENP1功能在何时何地对胚胎至关重要的见解。初步结果表明在颅面发育中的作用。完整的阐明将为SENP1和Sumoylation在发育过程中的生物学功能提供重要的新见解。我们还开发了一种方法来识别可能逃避基于差异mRNA表达的筛选策略的发育重要分子。泛素/蛋白酶体途径的快速蛋白质降解在许多关键的细胞过程中至关重要，包括细胞周期进程和程序性细胞死亡。我们假设发育过程中许多关键调控分子的活性受到泛素介导的蛋白质降解的调节，这些分子可以通过它们与E3泛素连接酶的相互作用来识别，泛素连接酶是泛素化途径中赋予特异性的组成部分。我们使用发育调控的E3 Nedd-4在妊娠中期表达的酵母双杂交筛选中分离出4个候选基因，Nedd-4 Binding Proteins (NBP) 1-4。其中三个是新基因，第四个编码PLIC2，一种介导CD47和细胞骨架之间相互作用的蛋白质。进一步的实验表明NBP1和-2在体外被ned4泛素化。我们有初步证据表明NBP2在体内也是泛素化的。最终，我们感兴趣的关键问题是NBP1-4是否对发育至关重要，特别是它们与泛素化过程的相互作用，包括快速降解的潜力，可能发挥什么作用。我们正在进行体外诱变，以确定NBP1-4与ned4结合的区域。阻止与ned4结合的突变可能使这些蛋白从泛素化过程中分离。

We are using two approaches to identify and isolate genes involved in the regulated growth and differentiation of the mammalian embryo and fetus. The first of these is insertional mutagenesis, in which mutant phenotypes are generated by the insertion of exogenous DNA into a gene. This also creates a molecular tag at the mutant locus, providing direct access to the gene. For several years, we have analyzed transgenic mouse strains derived from embryonic stem (ES) cells deliberately infected in tissue culture with retroviruses to generate insertional mutations. We screened over 40 proviral insertions specifically for mutations that lead to prenatal lethality, indicating that the gene is absolutely essential for normal development. This screen identified 4 recessive prenatal lethal mutations tightly linked to, and presumably caused by, proviral insertion. Currently we are focusing on the insertional mutation caused by the 412-r provirus. We isolated the 412-r retroviral insertion site and surrounding DNA in cosmids. Fragments of candidate genes were obtained using exon-trapping, extended by 5' and 3' RACE, and used for probing embryo cDNA libraries. These efforts allowed us to identify a candidate for the mutated gene. The provirus lies in the first intron of the gene for Sumo/sentrin specific protease 1 (SENP1), a recently identified protein involved in the regulation of Sumoylation. Sumo/sentrin is a small protein related to ubiquitin that is added to proteins in a process similar to ubiquitination. SENP1 acts to remove Sumo from modified proteins. Our evidence for 412-r mutating the SENP1 gene comes from the analysis of expression levels in homozygotes, which have an approximately 50% reduction. SENP1 is normally ubiquitously expressed. Either the viral insertion causes a reduced expression level in all cells or completely down-regulates the gene in only some cell or tissue types. Currently we are assessing this by in situ hybridization of mutant embryos. Our ongoing analysis of the 412-r mutant phenotype will provide insight into where and when SENP1 function is critically important for the embryo. Preliminary results suggest roles in craniofacial development. Complete elucidation will provide important new insights into the biological functions of SENP1 and Sumoylation during development. We also have developed an approach to identify developmentally important molecules that might elude screening strategies based on differential mRNA expression. Rapid protein degradation by the ubiquitin/proteasome pathway is of critical importance in many key cellular processes, including cell cycle progression and programmed cell death. We hypothesized that the activity of many critical regulatory molecules in development is regulated by ubiquitin-mediated protein degradation, and that these molecules can be identified by their interaction with E3 ubiquitin ligases, the component of the ubiquitination pathway conferring specificity. We used the developmentally regulated E3 Nedd-4 in a yeast two-hybrid screen of genes expressed at midgestation and isolated 4 candidates, Nedd-4 Binding Proteins (NBP) 1-4. Three of these are novel genes and the fourth encodes PLIC2, a protein that mediates interactions between CD47 and the cytoskeleton. Further experiments showed that NBP1 and -2 are ubiquitinated in vitro by Nedd-4. We have preliminary evidence that NBP2 is ubiquitinated in vivo as well. Ultimately, we are interested in the key question of whether NBP1-4 are crucial for development and specifically what role their interaction with the ubiquitination process, including the potential for rapid degradation, might play. We are carrying out in vitro mutagenesis to identify regions that confer binding of NBP1-4 to Nedd-4. Mutations that prevent binding to Nedd-4 may uncouple these proteins from the ubiquitination process.