Production of mouse models for human diseases by gene targeting

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。我们拜访了北卡罗来纳州立大学的Smithies博士，讨论了转基因技术的最新进展。通过将ES细胞与桑椹胚聚合而产生的嵌合体仅用于有限的细胞系，如Nagy开发的R1。现在，通过导入含有高达1兆碱基DNA的YAC DNA来生产转基因小鼠是可能的。含有多胺的溶液处理的YAC DNA的尺寸被证明小于用于将DNA显微注射到受精卵原核的注射吸管的内径。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型YAC DNA的分解，在提取DNA之前必须用琼脂糖酶完全溶解琼脂糖。我们访问了Max-Planck免疫生物学研究所的Solter博士，讨论了基因捕获方法。开发一种新的筛选系统来鉴定与芽胞形成有关的基因是很重要的。因此，我们在不含LIF的培养液中培养ES细胞。这些ES细胞在几天内聚集并发育成类胚体。在肝细胞分化过程中，肝特异表达基因，如转甲状腺素基因或白蛋白基因，也有类似的表达模式。噬菌体重组系统，Cre-loxP，被证明是有用的，以组织特异性和发育特异性的方式敲除该基因。还建议重组酶Cre在细胞核中表达，以有效地发挥其作用。

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.