DISRUPTION OF SPERMATID-SPECIFIC TBP EXPRESSION IN MICE

一些具有基本普遍作用的基因也表现出细胞类型- 具体功能。因为破坏无所不在的功能的突变 一般都是致命的，一直很难研究具体的作用 这样的基因。因此，从基因上操纵将是有用的 这些基因以定向的方式实现特定的功能。这个项目 研究一种基因的高度组织特异性功能，其产物 是所有真核细胞绝对需要的。与塔塔绑定 蛋白质，TBP，是所有核转录起始和 因此，对大多数细胞功能来说是至关重要的。在精子细胞中，TBP 基因的表达水平大约是那些 在体细胞中。这表明TBP在 精子发生。这个项目的目标是首先颠覆，然后 系统抢救，TBP的精子细胞特异性功能 转基因小鼠。因为所有的核转录都需要TBP， 重要的是，正常的体细胞表达和功能不会 打乱了。为了实现这一目标，将采取两种不同的方法。 首先，噬菌体P1Cre/loxP位点特异性重组 系统将被用来精确地切除睾丸特异性启动子/ 第一外显子，而不破坏体细胞启动子/第一外显子。在一个 另一种方法，所有交替的TBP基因启动子/第一外显子 将被移除并被编码该基因的5‘端区域所取代 普遍存在的转录因子，Oct1。Oct1基因的表达水平为 在数量上与体细胞组织中的TBP mRNA水平相似，但 在睾丸中不过度表达。后一种方法可能会被证明是有价值的 如果在前一种方法中，剩余启动子/的过度表达 第一外显子补偿精子细胞特异性转录的丢失 单位。在精子细胞中不能过表达TBP的转基因小鼠系 将用于补充实验。携带野性的病媒 TBP基因的类型或体外修饰版本将在 突变的生殖系，以确定TBP的哪些结构域必须完整 外源蛋白来挽救精子发生。这应该是 深入了解TBP在精子细胞中的特定作用，并提供 一个有价值的范例，说明基本基因的特定活动如何 有选择地被扰乱。

Some genes with fundamental ubiquitous roles also exhibit cell type- specific functions. Since mutations that disrupt ubiquitous function are generally lethal, it has been difficult to study the specific roles of such genes. Thus, it would be useful to genetically manipulate specific functions of such genes in a directed fashion. This project investigates a highly tissue-specific function of a gene whose product is absolutely required by all eucaryotic cells. The TATA-binding protein, TBP, is required for all nuclear transcription initiation and thus is crucial for most cellular functions. In spermatids, the tbp gene is expressed at levels about 100- to 1000-fold greater than those in somatic cells. This suggests that TBP plays a specialized role in spermatogenesis. The goal of this project is to first disrupt, and then systematically rescue, the spermatid-specific function of TBP in transgenic mice. Because TBP is required for all nuclear transcription, it is important that normal somatic cell expression and function not be disrupted. To achieve this, two different approaches will be taken. In the first, the bacteriophage P1 Cre/loxP site-specific recombination system will be used to precisely excise the testis-specific promoter/ first exons without disrupting the somatic promoter/first exon. In an alternate approach, all of the alternate tbp gene promoter/first exons will be removed and replaced with the 5' region of the gene encoding the ubiquitous transcription factor, Oct1. Oct1 mRNA levels are quantitatively similar to TBP mRNA levels in somatic tissues, but are not overexpressed in testis. This later approach might prove valuable if, in the former approach, overexpression of the remaining promoter/ first exon compensates for loss of the spermatid-specific transcription units. Transgenic mouse lines that fail to overexpress TBP in spermatids will be used for complementation experiments. Vectors carrying wild type or in vitro-modified versions of the tbp gene will be expressed in the mutant germ lines to determine what domains of TBP must be intact for the exogenous protein to rescue spermatogenesis. This should provide insights into the spermatid-specific roles of TBP and provide a valuable paradigm for how specific activities of fundamental genes can be selectively disrupted.