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

1.卵母细胞体外成熟和受精：为了建立最佳的配子和胚胎操作条件，对影响卵母细胞体外成熟和受精的各种因素进行了研究。我们发现在猪卵泡液中成熟的猪卵母细胞具有正常的发育能力，它们在减数分裂成熟过程中表现出基本相同的组蛋白H1激酶活性的波动规律。研究还发现，提高受精液中钙离子浓度可以显著提高去卵丘小鼠卵母细胞的体外受精率。携带SV40早期启动子与大肠杆菌β-半乳糖苷酶基因融合的DNA(LacZ)显微注射入体外受精卵的原核，X-Gal染色检测其表达。结果表明，外源基因从4-细胞期开始表达，在桑蚕期达到最大值。所有胚胎都显示出马赛克染色图案，这表明注入的DNA上分布不均和/或卵裂球的早期分化。建立新的ES细胞系：我们从129/SVJ小鼠的囊胚中建立了两个ES细胞系。来自一个细胞系(A3-1)的细胞被证明具有多能性，包括注射到宿主囊胚后发育成生殖系嵌合体的能力。这些细胞具有XY性染色体，白化基因座为纯合子。我们进一步发现，将胚泡培养过夜孵化，然后转移到饲养层细胞上，分离ES细胞的效率明显提高。

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.