Production of mouse models for human diseases by gene targeting

本项目旨在探讨基因靶向技术在生物医学研究中的可能性及其应用。我们拜访了北卡罗莱纳州立大学的史密斯博士，讨论了转基因技术的最新进展。将胚胎干细胞与桑葚胚聚集在一起产生嵌合体的方法仅用于有限的细胞系，如Nagy开发的R1。现在有可能通过引入含有高达1兆碱基DNA的YAC DNA来生产转基因小鼠。经多胺溶液处理后的YAC DNA的大小小于用于向受精卵原核微量注射DNA的注射管的内径。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之前必须用琼脂酶完全溶解琼脂糖。我们拜访了马克斯-普朗克免疫生物学研究所的solter博士，讨论了基因诱捕法。建立一种新的筛选系统来鉴定参与胚层形成的基因具有重要的意义。因此，我们在不含liff的培养基中培养胚胎干细胞。这些胚胎干细胞在几天内聚集并发育成胚状体。肝脏特异性基因，如转甲状腺素基因或白蛋白基因，在肝细胞分化过程中以类似的模式表达。噬菌体重组系统Cre-loxP被证明能够以组织特异性和发育特异性的方式敲除该基因。也有人认为重组酶Cre应该在细胞核中表达才能有效发挥其作用。

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.