Studies on the Gene Expression Mcchanisms as the Basis for the Development of Industrially Usuful Transgenic Animals

基因表达机制的研究作为开发工业上有用的转基因动物的基础

基本信息

批准号：
05454111
负责人：
TACHI Chikashi
金额：
$ 4.48万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

During the period of research, following mjor results were obtained. 1)A genomic DNA library of Shiba goat (Capra hircus var Shiba) was constructed using lambda phage as the vector. The library will serve as an important source of industrially useful genes of artiodactylas. 2)Caprine SRY gene was sucssessfully cloned complete with the promoter region and 3'untranaslated region. The gene will be potentially useful in developing the means for the control of sex ratios at birth in farm animals. 3)murine Sry-transfected cell lines where the linear functional Sry transcripts were expressed, were established using a Sertoli cell-derived cell line, TM-4, and an ES cell line with XX karyotype, TMA-18, as tools for the study of SRY action within cells. In the TM-4 derived Sry-transfected cell lines, P450 aromatase gene expression was induced dc novo as a results of unscheduled expression of Sry. On the other hand, WT1 (Wilm's tumor suppressor gene 1 was mobilized by the Sry transgene expression … More . 4)Tissue specificity of murine WAP (whey acidic protein) promoter was analyzed in vivo using RT-PCR method. It was found that WAP gene expression was not limited to the mammary gland, as has generally been believed to be, and is of rather ubiquitous nature. The finding will have important bearings upon the usufulness of WAP promoter in directing the transgene expression to the mammary gland. 5)Effect of over-expression of the long-form prolactin receptor gene, PRLR_L upon the expression of prolactin-target genes coding for beta-casein and WAP,was investigated by transfecting HC11 cells, a cell line established from a mammary gland of pregnant BALB/c mouse, with a DNA construct where PRLR_L cDNA was fused with the MMTV-promoter. Surprisingly, the results indicated that the over-expression of the receptor lead to the down-regulation rather than the up-regulation of the target genes. Presumably, the competition for the signal transduction molecules has occured and a new model, "an abortive hit model" was developed. 6)Complete sequence of the caprine c-kit cDNA was determined. It was revealed that the KI domain of the caprine c-kit possesses a novel alanine insert. Comaparative analysis of the aa sequence of the KI domain in different species of mammals confirmed that the insert is specific to goats and sheep. A computer simulation model, Robson-plot, predicted that the alanin insert in the KI domain cause a considerable change in the secondary structure of the peptide in the region, possibly leading to the functional modification of the c-kit in those species. Less

在研究期间，获得了MJOR结果。 1）使用lambda噬菌体作为载体构建了shiba山羊（Capra hircus var shiba）的基因组DNA文库。该图书馆将成为artiodactylas的工业有用基因的重要来源。 2）caprine sry基因被用启动子区域和3'untranaslated区域进行了克隆。该基因将在开发农场动物出生时控制性别比的手段方面有可能有用。 3）用Sertoli细胞衍生的细胞系TM-4和带有XX Karyotype，TMA-18的ES细胞系来确定线性功能性SRY转录本的鼠SRY转染细胞系，作为用于研究细胞内SRY动作的工具。在TM-4衍生的SRY转染细胞系中，p450芳香酶基因表达是由于SRY的外表达而引起的DC NovO。另一方面，WT1（WILM的肿瘤抑制基因1通过SRY转基因表达动员……更多。4）使用RT-PCR方法在体内分析鼠WAP（乳清酸性蛋白）启动子的组织特异性。已经发现，正如通常认为的那样，WAP基因表达不限于乳腺，并且具有无处不在的性质。 The finding will have important bearings upon the usufulness of WAP promoter in 5) Effect of over-expression of the long-form prolactin receptor gene, PRLR_L upon the expression of prolactin-target genes coding for beta-casein and WAP, was investigated by transfecting HC11 cells, a cell line established from a mammary gland of pregnant BALB/c mouse, with a DNA construct where PRLR_L cDNA was fused与MMTV启动器。令人惊讶的是，结果表明受体的过表达导致下调，而不是目标基因的上调。据推测，已经发生了信号转导分子的竞争，并且开发了一种新模型，即“流产命中模型”。 6）确定了胶囊C-KIT cDNA的完整序列。据透露，Caprine C-Kit的Ki域具有新颖的丙氨酸插入物。对不同哺乳动物中Ki结构域的AA序列的分析证实，该插入物特定于山羊和绵羊。一个计算机模拟模型Robson-Plot预测，Ki域中的alanin插入物会导致该区域肽的二级结构的考虑变化，这可能导致该物种中C-KIT的功能修饰。较少的