Basic studies on production and application of transgenic animals in ruminants

1.从具有显性黑眼白色表型的芝山羊（Capra hircus var Shiba）中克隆了c-kit cDNA的完整开放阅读序列。推导的氨基酸序列的分析揭示了一个单一的氨基酸插入（丙氨酸）的激酶插入区域的存在。2.研究了小鼠和山羊胎盘组织中同源异型盒基因的表达。在小鼠胎盘中，除了Hox c9外，所有Hox 9旁系同源物都表达。在山羊胎盘中，除了HOX d9外，还检测到HOX 9旁系同源物。3.人们试图研究将外源DNA构建体与细菌限制性内切酶共注射到小鼠受精卵的原核中是否能够提高外源DNA整合到宿主基因组中的频率。结果表明，限制性内切酶共注射基因在胚胎和幼仔中的整合成功率略有提高。4.提高转基因动物生产效率的一种方法是在将转基因胚胎转移到寄养母亲体内之前对其进行鉴定。绿色荧光蛋白（GFP）可作为转基因胚胎筛选的合适标记。将CMV beta-actin/EGFP基因显微注射入小鼠受精卵原核。将注射DNA的胚胎进行体外培养并观察荧光。将荧光胚胎移植到受体小鼠的子宫中。对出生的幼崽进行转基因分析。用Dpn I-Bal 31消化法对部分胚胎进行了转基因分析。结果表明，约77%的具有均匀荧光的胚胎是转基因的。本研究表明，以EGEP为标记的转基因胚胎筛选方法是非常可靠的。

1. The complete open reading tram of the c-kit cDNA was clone from the Shiba goat (Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanin) in the kinase insert region. 2. The expressions of homeobox genes in murine and caprine placentae were surveyed. In the murine placentae, all the Hox 9 paralogues except for Hox c9 were expressed. In the caprine placentae, the HOX 9 paralogues except for HOX d9 were detected. 3. Attempts were made to investigate whether co-injection of foreign DNA constructs with bacterial restriction endonuciease into the pronucleus of mouse zygotes will be capable of elevating integration frequency of foreign DNA into the host genome. The results showed that co-injection of the genes with restriction enzyme appeared the slight elevation of successful integration in the embryos and the pups. 4. One way to improve the efficiency of transgenic animal production would be to identify the transgenic embryos before transfer into foster mothers. Green fluorescent protein (GFP) can be a suitable marker for the selection of transgenic embryos. CMV beta-actin/EGFP gene was microinjected into the pronucleus of mouse zygotes. The DNA injected embryos were cultured in vitro and observed for fluorescence. The fluorescent embryos were transferred into the uteri of recipient mice. Pups born were analyzed for transgenic. A part of the embryos were analyzed for the transgene by the Dpn I-Bal31 digestion method. The results showed that approximately 77% of the embryos with uniform fluorescence were transgenic. The present study demonstrated that the method using EGEP as a marker be very reliable for selecting the transgenic embryos.