Analysis of the function of the reproductive and immune system an approach by the embryo manipulation

为了研究转基因小鼠的生殖和免疫功能，将编码小鼠金属硫蛋白调控区和人生长激素结构基因的线性DNA片段微注射到C57BL/6卵细胞的雄性原核中。注射后，卵培养过夜，移植到假孕输卵管中。用斑点杂交和北方杂交的方法，在一只活产小鼠的基因组DNA中检测到大约150个人类生长激素基因拷贝。培养、着床、出生成活率正常，但融合率仍较低。为了利用转基因小鼠作为蛋白质合成系统，我们克隆了小鼠酪蛋白44k和乳清酸性蛋白的cDNA。这一系列的研究还表明，除了输卵管因子外，细胞质因子的缺失也参与了“双细胞阻滞”。此外，还制备了具有与人类菌群相似菌群的生物小鼠，以分析其对免疫系统和饮食系统的影响。由于催乳素是调节动物生殖和免疫功能的关键激素，催乳素的作用方式是本课题的主要研究对象之一。巨噬细胞参与免疫系统和生殖系统的调节。从兔乳腺中部分纯化催乳素受体，制备单克隆抗体。我们获得了两个识别受体并抑制催乳素结合的克隆。使用这些抗体存在两类受体位点。在大鼠黄体中，催乳素抑制20< α >-羟基类固醇脱氢酶的活性，该酶也被部分纯化。该酶制剂可用于阐明催乳素在黄体中的促黄体作用模式。在未来，转基因小鼠和非生物小鼠将被用作分析生殖和免疫系统的工具。

To investigate the reproductive and immune functions of transgenic mice, a linear DNA fragment encoding the regulatory region of mouse metalothionein and the strucural gene of human growth hormone was microinjected into the male pronucleus of C57BL/6 ova. After injection the ova were cultured overnight and transplanted into pseudopregnant oviducts. About 150 copies of human growth hormone gene was detected by the dot-blot gybridization and northern hybridization in the genomic DNA of a mouse born alive. The rates of survival in culture, implantation, and birth were normal, but the rate of integration was still low. For the use of transgenic mice as a protein synthetic system, we cloned cDNA for mouse casein 44k and whey acidic protein. It was also suggested in this series of study that the absence of a cytoplasmic factor or factors is involved in "two-cell block" in addition to an oviduct factor as has been suggested. Gnotobiote mice were also produced which have flora similar to the human flora to analyze their effects on the immune and dietary systems. The mode of action of prolactin is one the main subjects to study in this program because prolactin is a key hormone which regulates reproductive and immune function of animals. Macrophages are involved in the regulation of both immune and reproductive systems. Prolactin receptor was partially purified from rabbit mammary glands, and monoclonal antibodies were raised against it. We obtained two clones which recognized the recepto and inhibited prolactin binding. Using these antibodies the presence of two classes of receptor sites was suggested. In rat corpora lutea, prolactin inhibits the activity of 20<alpha>-hydroxysteroid dehydrogenase, which also was partially purified. This enzyme preparation can be used to clarified the mode of luteotrophic action of prolactin in corpora lutea. Transgenic mice and gnotobiote mice will be used as tools to anlyze the reproductive and immune systems in future.