Manipulation of mdx mouse embryos

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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.