Basic study on the production of transgenic animals through gametes and embryonic stem cells

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1. Oocyte maturation and fertilization in vitro.In order to establish the optimum condition for manipulating gametes and embryos, various factors affecting oocyte maturation and fertilization were investigated in vitro. We have found that porcine oocytes matured in porcine follicular fluid have normal developmental ability and they showed essentially the same fluctuation pattern of histone Hl kinase activity during the course of meiotic maturation. It was also found that the in vitro fertilization of cumulus-free mouse oocytes was improved significantly by increasing calcium concentration in the fertilization medium.2. Expression of exogenous genes in preimplantation embryos.DNA carrying the SV40 early promoter fused with the Escherichia coli beta-galactosidase gene, (lacZ) was microinjected into the pronucleus of in vitro fertilized mouse eggs and the expression was detected by staining embryos with X-gal. It was found that the exogenous gene was expressed from the 4-cell stage and reached the maximum at the morula stage. All of the embryos have shown a mosaic pattern of staining, suggesting unequal distribution on injected DNA and/or early differentiation of blastomeres.3. Establishment of new ES cell lines.We have established two ES cell lines from the blastocysts of 129/SvJ mice. The cells from one cell line (A3-1) were demonstrated to have pluripotency, including the ability to develop to germ line chimeras following injection into the host blastocysts. The cells have XY sex chromosomes and are homozygous for the albino locus. We further found that the efficiency of isolation of ES cells was markedly improved when the blastocysts were cultured overnight for hatching and then transferred on the feeder cells.