Is Basonuclin essential for Oogenesis

描述(申请人提供)：Basonuclin是一种120 kDa的锌指蛋白，可能是一种转录因子，可以增强核糖体RNA(RRNA)基因的转录。它的表达仅限于睾丸和卵巢的生殖细胞，以及复层上皮的角质形成细胞。长期以来，人们一直认为rRNA基因转录是决定核糖体产生的关键因素，它受细胞蛋白质合成水平的调节。某些细胞生理状态，如增殖(精子发生)或蛋白质积累(卵子发生)，需要高水平的蛋白质合成。虽然已经描述了与增殖过程中高水平的核糖体合成相关的转录因子，但这些因子似乎无处不在，而rRNA合成的细胞类型特异性调节因子尚未见报道。Basonuclin由于其(1)限制细胞类型分布，(2)与间期和有丝分裂中的rRNA基因有关，(3)与rRNA基因的启动子相互作用，以及(4)从rDNA启动子增加转录的能力，很可能是一种细胞型特异的rRNA转录因子，这是第一个此类转录因子；(5)我们用常规技术敲除basonuclin基因的尝试没有成功，因为零突变不能传递到生殖系，这表明basonuclin单倍体不足以促进生殖细胞的发育。此外，我们最近的研究结果表明，Basonuclin在小鼠卵子发生过程中发挥着关键作用。基于这些观察，我们推测Basonuclin在小鼠早期卵子发生的核糖体生物发生中是必不可少的。如果是这样的话，抑制或有针对性地破坏卵母细胞中碱性核蛋白的功能应该会扰乱它的发育，或者从它衍生的胚胎的发育。由于我们早先未能通过传统技术创建Basonuelin基因敲除，在这一应用中，我们建议以单一的特定目标来测试采用两种最近开发的条件基因干扰技术的可行性。SubAim 1将使用RNAi方法减少卵母细胞中Basonuclin的数量；Sub Aim 2将使用Cre重组酶敲除卵母细胞中的Basonuclin基因。

DESCRIPTION (provided by applicant): Basonuclin, a zinc finger protein of 120 kDa, is likely to be a transcription factor that enhances transcription of ribosomal RNA (rRNA) genes. Its expression is restricted to the germ cells of the testis and ovary, and keratinocytes of the stratified epithelium. It has been long recognized that rRNA gene transcription, a key factor in determining ribosome production, is regulated according to the level of cellular protein synthesis. Certain cellular physiological states, such as proliferation (spermatogenesis) or protein accumulation (oogenesis) require a high level of protein synthesis. Although transcription factors associated with the high level of ribosome synthesis during proliferation have been described, these factors appear to be ubiquitous, whereas cell-type specific regulators of rRNA synthesis have not been reported. Basonuclin, because of its: (1) restricted cell-type distribution, (2) association with the rRNA genes in interphase as well as in mitosis, (3) interaction with the promoter of the rRNA genes, and (4) ability to increase transcription from an rDNA promtor, is likely to be a cell-type specific rRNA transcription factor, the first of its kind; (5) our attempt to knockout basonuclin gene by conventional technique was not successful because the null mutation cannot be transmitted into the germline, suggesting that basonuclin is haploinsufficient for germ cell development. Furthermore, the results of our recent study suggest that basonuclin plays a critical role during mouse oogenesis. Based on these observations, we hypothesize that basonuclin is essential in ribosome biogenesis during early mouse oogenesis. If so, inhibition, or targeted disruption, of the function of basonuclin in an oocyte should perturb its development, or the development of the embryo derived from it. Because of our earlier failure in creating a basonuelin knockout by conventional techniques, in this application, we propose, in a single Specific Aim, to test the feasibility of employing two recently developed conditional gene-disruption techniques. Sub aim 1 will use an RNAi approach to reduce the amount of basonuclin in oocytes; Sub aim 2 will use the Cre recombinase to knockout [the] basonuclin gene in oocytes.