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Production of mouse models for human diseases by gene targeting

通过基因打靶制作人类疾病小鼠模型

基本信息

批准号：
04044136
负责人：
YAMAMURA Ken-ichi
金额：
$ 3.78万
依托单位：
Institute of Molecular Embryology and Genetics Kumamoto University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

The purpose of this project is to discuss about the possibility and application of gene targeting techniques in biomedical research. We visited Dr.Smithies, North Carolina State University, to discuss about the recent advances in transgenic technology. Chimera production by aggregating ES cells with morula was shown to be used for only limited cell line, such as R1 developed by Nagy. It is now possible to produce transgenic mice by introducing YAC DNA containing up to 1 megabase DNA.The size of YAC DNA treated with solution containing polyamine was demonstrated to be smaller than the inner diameter of injection pipette used for microinjection of DNA into pronuclei of fertilized eggs. Thus, the microinjection method was most feasible approach to produce YAC transgenic mice. As the contamination of agarose results in the breakdown of giant YAC DNA,it is essential to dissolve agarose completely by agarase before isolation of DNA.We visited Dr.Solter, Max-Planck-Institute for lmmunobiology, to discuss about the gene trap method. It is important to develop a new screening system to identify the genes involved in the formation of germ layr. Thus, we cultured ES cells in LIF-free medium. These ES Cells aggregated within a few days and developed into embryoid body. Liver-specific genes, such as transthyretin gene or, albumin gene, were expressed in a similar pattern observed during liver cell differentiation. Bacteriophage recombination system, Cre-loxP,is shown to be useful to knock out the gene in a tissue-specific and developmentally specific manner. It was also suggested that the recombinase, Cre, should be expressed in a nucleus to exert its effect efficiently.

本项目的目的是探讨基因打靶技术在生物医学研究中的可能性和应用。我们访问了北卡罗来纳州州立大学的史密斯博士，讨论了转基因技术的最新进展。通过ES细胞与桑椹胚聚集的嵌合体生产显示仅用于有限的细胞系，如Nagy开发的R1。用多胺溶液处理的YAC DNA，其大小比显微注射DNA到受精卵原核的注射管内径小。因此，显微注射法是制备YAC转基因小鼠最可行的方法。由于琼脂糖的污染会导致巨大的YAC DNA的分解，因此在分离DNA之前必须用琼脂糖酶完全溶解琼脂糖。因此，建立一种新的筛选体系，对参与胚层形成的基因进行筛选具有重要意义。因此，我们在无LIF培养基中培养ES细胞。这些ES细胞在几天内聚集并发育成胚状体。肝特异性基因，如甲状腺素运载蛋白基因或白蛋白基因，在肝细胞分化过程中观察到类似的模式表达。噬菌体重组系统，Cre-loxP，被证明是有用的敲除基因的组织特异性和发育特异性的方式。还建议重组酶Cre应在细胞核中表达以有效地发挥其作用。