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

为了制备大鼠生长激素（GH）基因序列互补的反义DNA转基因大鼠，并探讨反义RNA表达对GH基因表达和生长发育的调节作用，我们将一个融合基因导入大鼠胚胎，由含有四个拷贝甲状腺激素反应元件（TRE）的大鼠GH启动子组成该基因启动子可使CAT基因的表达增强2.8倍。4只转基因大鼠中有2只在垂体和淋巴结中表达较强的反义RNA，每只建立者大鼠的转基因后代（F_1）早在3-4周龄就表现出侏儒症，与非转基因同窝出生的小鼠相比，它们的生长速度降低了70-80%。在转基因大鼠中特异性表达反义GH RNA并调节生长发育，当将大鼠GH反义cDNA单独连接到大鼠GH启动子时，在大鼠胚胎中，转基因fonder大鼠的后代（F1）没有表现出侏儒症，然而使用RT-PCR在它们的各种组织包括垂体和淋巴组织中检测到反义RNA的表达。我们正在尝试通过导入反义RNA来抑制GH基因的表达，从而培育小型山羊，我们还证明了人甲状腺素运载蛋白基因的上游6-kb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。

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.