Production of a Germ-line Chimera by Coculture of Zona-free Embryos with Frozen-thawed Embryonic Stem Cells.

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Abstract
To improve chimera production efficiency, we examined whether frozen-thawed embry-onic stem (ES) cells would be useful in a coculture method. A3-1 ES cells were electroporated with the targeting vector pE 10.29NEO-TK. Exon 2 of the endothelin-1 gene in the ES cells was disrupted by homologous recombination. The homologous recombinant was cocultured at a density of 5.5 × 105 cells/ml with zona-free 8-cell to morula stage embryos in a coculture medium for 3-3.5 h. Cocultured embryos were transferred into Whitten's medium and cultured overnight. Morula and blastocyst stage embryos were transferred into the recipients on day 2.5 of pseudopregnancy. The development rate to blastocyst after coculture and subsequent overnight cultivation was 62%. As a result of embryo transfer, four male chimeras were obtained. Two of these were identified as germ-line chimeras by a progeny test. These results indicate that frozen-thawed ES cells can be used in coculture to produce chimeras and contribute a germ-line.

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