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Production of transgenic livestock animals bearing antisense growth hormone cDNA and analysis of mechanisms of the transgene expression

反义生长激素cDNA转基因家畜的制备及转基因表达机制分析

基本信息

批准号：
05304022
负责人：
YAMADA Masayasu
金额：
$ 3.52万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
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

In an attempt to produce transgenic rats bearing antisense DNA complementary to sequences in the rat growth hormone(GH)gege and to investigate whether antisense RNA expression can modulate GH gene expression and body growth , we produced four transgenic founder rats(F_0)by introducing into rat embryos a fusion gene, which consisted of rat GH promoter containing with four copies of thyroid hormone response element(TRE)and antisense cDNA sequences for rat GH.This transgene promoter directed 2.8-fold stimulation of CAT gene expression in transfected GH_3 rat pituitary tumor cells compared with the rat GH promoter alone.Two of four transgenic rats expressed strongly antisense RNA in the pituitary and lymphoid tissues.Transgenicoffspring(F_1)from each founder rat exhibited dwarfism at as early as 3-4 weeks of age, and they exhibited 70-80% reduced growth rate compared with their nontransgenic littermates.We next examined the effects of inserting tandem copies of TRE on tissue-specific expression of antisense GH RNA and modulation of body growth in the transgenic rats.When antisense cDNA for rat GH linked to rat GH promoter alone was introduced into rat embryos, the offspring(F1)of the transgenic fonder rats did not exhibited dwarfism, whereas expression of antisense RNA was detected in their various tissues including pituitary and lymphoid tissues using RT-PCR.From the results, it is suggested that TRE is necessary for the suppression of GH gene expression by antisense RNA.We are now trying to produce a miniature goat by inhibiting GH gene expresion with introduction of antisense goat GH cDNA into pronuclear embryos derived from IVM/IVF oocytes.We have also demonstrated that a 6-kb upstream region of the human transthyretin gene retained all the cis-elements required for developmental, tissue-specific, and quantitatively normal expression.

为了获得与大鼠生长激素（GH）基因序列互补的反义DNA转基因大鼠，并研究反义RNA表达是否能调节GH基因的表达和机体生长，我们将含有4拷贝甲状腺激素应答元件（TRE）的大鼠GH启动子和大鼠生长激素反义cDNA序列的融合基因导入大鼠胚胎，获得了4只转基因奠基人大鼠（F_0）。该转基因启动子对转染GH_3的大鼠垂体瘤细胞CAT基因表达的刺激作用是单独使用GH启动子的2.8倍。4只转基因大鼠中有2只在垂体和淋巴组织中表达强烈的反义RNA。每只创始鼠的转基因后代（F_1）早在3-4周龄时就表现出侏儒症，其生长速度比非转基因幼鼠低70-80%。接下来，我们研究了插入串联TRE拷贝对反义GH RNA的组织特异性表达和转基因大鼠身体生长调节的影响。将单独与大鼠生长激素启动子相关的大鼠生长激素反义cDNA导入大鼠胚胎后，转基因幼鼠后代（F1）未出现侏儒症，而RT-PCR检测到反义RNA在其垂体和淋巴组织等多种组织中表达。结果表明，TRE是反义RNA抑制生长激素基因表达所必需的。我们现在正试图通过将反义山羊生长激素cDNA引入来自IVM/IVF卵母细胞的原核胚胎中来抑制生长激素基因的表达，从而生产出小型山羊。我们还证明，人甲状腺转甲素基因的上游6kb区域保留了发育、组织特异性和定量正常表达所需的所有顺式元件。