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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

在研究过程中，取得了以下主要成果。1）以λ噬菌体为载体，构建了芝山羊（Capra hircus var Shiba）基因组DNA文库。该文库将作为偶蹄目动物工业有用基因的重要来源。2)成功地克隆了山羊SRY基因的启动子区和3 '端非转录区。该基因将在开发控制家畜出生性别比的方法方面具有潜在的用途。3)使用Sertoli细胞衍生的细胞系TM-4和具有XX核型的ES细胞系TMA-18作为研究细胞内SRY作用的工具，建立表达线性功能性Sry转录物的鼠Sry转染细胞系。在TM-4衍生的Sry转染细胞系中，作为Sry的非预定表达的结果，P450芳香化酶基因表达被从头诱导。另一方面，WT 1（Wilm肿瘤抑制基因1）通过Sry转基因表达被动员 ...更多信息 . 4)采用RT-PCR方法分析了小鼠WAP（乳清酸性蛋白）启动子的体内组织特异性。结果发现，WAP基因表达并不局限于乳腺，一般认为是，是相当普遍的性质。这一发现将对WAP启动子在乳腺中指导转基因表达具有重要意义。5)通过将长型催乳素受体基因（PRLR_L）cDNA与MMTV启动子融合，构建了一个DNA构建体，并转染孕鼠乳腺细胞系HC 11，研究了PRLR_L过表达对催乳素靶基因β-酪蛋白和WAP表达的影响。令人惊讶的是，结果表明受体的过表达导致靶基因的下调而不是上调。推测这一过程中可能发生了对信号转导分子的竞争，并发展了一个新的模型--“一个失败的击中模型”。6)测定了山羊c-kit cDNA的全序列。结果表明，山羊c-kit的KI结构域具有一个新的丙氨酸插入。对不同哺乳动物KI结构域氨基酸序列的比较分析证实，该插入片段对山羊和绵羊具有特异性。计算机模拟模型Robson-plot预测，KI结构域中的丙氨酸插入导致该区域肽的二级结构发生相当大的变化，可能导致这些物种中c-kit的功能修饰。少