Manipulation of mdx mouse embryos

1)从mdx小鼠体内分离胚胎干细胞。收集mdx小鼠囊胚期胚胎，进行体外培养。取ICM来源的细胞团，经胰酶处理后转移到丝裂霉素处理的饲养层细胞上。分离并建立了稳定的未分化干细胞生长克隆。2)基因打靶技术我们已经打乱了等位基因N-myc。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。灭活的N-myc等位基因通过小鼠生殖系传播。3)转基因在小鼠嵌合体分析中的应用我们在小鼠胚胎中引入了外源基因，以便将转基因用作小鼠嵌合体的细胞标记。所选择的转基因为p321CAT，它含有与人延伸因子EF1pha启动子区域相连的CAT基因。首先，将该载体导入胚胎干细胞系，检测CAT基因的表达情况。选择其中一个稳定的转化子并用于生产小鼠嵌合体。用抗CAT抗血清免疫组织化学方法检测了宫内发育的小鼠嵌合体中CAT的活性。结果表明，在受精卵发育过程中，p321CAT基因在不同组织中普遍表达，表明p321CAT是一个有用的遗传标记。在其中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对小鼠嵌合体的分析非常有用。

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.