Manipulation of mdx mouse embryos

本研究建立了mdx小鼠胚胎操作技术，为mdx小鼠的进一步研究奠定了基础。1）mdx小鼠胚胎干细胞的分离：取mdx小鼠囊胚期胚胎，进行体外培养。将ICM衍生的细胞团块拾取，通过胰蛋白酶处理解聚，并转移到丝裂霉素处理的饲养细胞上。2）基因打靶技术我们已经破坏了等位基因N-myc基因。在ES细胞中通过携带新霉素抗性基因的靶向载体的同源重组。失活的N-myc等位基因通过小鼠生殖系传播。3）利用转基因分析小鼠嵌合体我们将外源基因导入小鼠胚胎中，以利用转基因作为小鼠嵌合体的细胞标记。选择的转基因是质粒p321 CAT，其含有与人延伸因子EF 1 α的启动子区连接的CAT基因。首先，将该质粒转入胚胎干细胞系中，检测CAT基因的表达。选择稳定的转化体之一并用于产生小鼠嵌合体。用CAT抗血清对子宫内发育的小鼠嵌合体进行了CAT活性的免疫组化检测，结果表明CAT基因在整个发育过程中在各种组织中普遍表达，提示质粒p321 CAT是一个有用的遗传标记。我们试图通过将质粒注射到受精小鼠卵的原核中来产生携带p321 CAT的转基因小鼠。在一只这样的转基因小鼠中，发现CAT基因普遍表达。这些转基因小鼠被发现是非常有用的小鼠嵌合体的分析。

We have developed embryo manipulating techniques in order to analyze mdxmice in future.1) Isolation of embryonic stem cells from mdx mice.The blastocyst-stage embryos of mdx mice were collected, cultured in vitro. The ICM-derived cell clumps were picked up, disaggregated by trypsin treatment and transferred on the mitomysin-treated feeder cells. Growing colonies of undifferentiated stem cells were isolated and established as a stable cell lines.2) Gene targeting techniqueWe have disrupted the allelic N-myc gene. in ES cells by means of homologous recombination of targeting vectors that carry neomycin resistant gene. The inactivated N-myc alleles were transmitted through mouse germ lines. The technique developed here will be useful to produce dystrophin-lacking new mouse strains in future.3) Use of a transgene for the analysis of mouse chimerasWe have introduced a foreign gene into mouse embryos in order to use the transgene as a cellular marker in mouse chimeras. The transgene chosen was a plasmid p321CAT which contains the CAT gene linked to the promoter region of human elongation factor EF1alpha. At first, The plasmid was transferred into embryonic stem cell lines and the expression of CAT gene was examined. One of the stable transformants was chosen and employed to produce mouse chimeras. The CAT activity in developing mouse chimeras in utero was examined immunohitochemically using an antiserum against CAT. The results show the CAT gene was expressed in various tissues ubiquitously through the course of development, suggesting the plasmid p321CAT is a useful genetic marker.Based on these results, we have attempted to produce a transgenic mouse carrying the p321 CAT by injecting the plasmid into pronuclei of fertilized mouse eggs. In one of such transgenic mice produced, the CAT gene was found to express ubiquitously. These transgenic mice are found to be quite useful for the analysis of mouse chimeras.