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

在研究期间，取得了以下研究成果。1)以Lambda噬菌体为载体，构建了山羊基因组DNA文库。该文库将成为偶蹄目动物工业有用基因的重要来源。2)成功克隆了山羊SRY基因的启动子区域和3‘端非翻译区。该基因可能有助于开发控制农场动物出生时性别比例的方法。3)以支持细胞来源的细胞系TM-4和XX核型的ES细胞系TMA-18为研究工具，建立了表达线性功能Sry转录本的Sry转基因小鼠细胞系。在TM-4来源的Sry转基因细胞系中，由于Sry的非程序性表达，P450芳香化酶基因的表达被DC novo诱导。另一方面，野生型肿瘤抑制基因1(Wt1)被Sry转基因表达的…激活更多。4)用RT-PCR方法分析了小鼠乳清酸性蛋白(WAP)启动子的体内组织特异性。研究发现，WAP基因的表达并不像人们通常认为的那样局限于乳腺，而且具有相当普遍的性质。这一发现将对WAP启动子将转基因表达定向到乳腺具有重要意义。5)将长型催乳素受体基因prlr_L与mmtv启动子融合后，通过转染孕鼠乳腺癌细胞系hc11细胞，研究了PRLR_L基因的过表达对β-酪蛋白和WAP基因表达的影响。令人惊讶的是，结果表明，受体的过度表达导致了靶基因的下调而不是上调。据推测，已经发生了对信号转导分子的竞争，并发展了一种新的模型--“流产的HIT模型”。6)测定了山羊c-kit基因的全序列。结果表明，山羊c-kit的KI结构域含有一个新的丙氨酸插入物。对不同物种哺乳动物Ki结构域的AA序列的比较分析证实，该插入片段是山羊和绵羊所特有的。计算机模拟模型Robson-Plot预测，在KI结构域插入丙氨酸会导致该区域多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。较少

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