Production of mouse models for human diseases by gene targeting

点击翻译按钮获取中文摘要

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.