Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations
Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations
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DOI：
10.1016/s0093-691x(02)01294-3
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发表时间：
2003-05-01
期刊：
影响因子：
2.8
通讯作者：
Hwang, WS
中科院分区：
文献类型：
--
作者：
Lee, GS;Hyun, SH;Hwang, WS
本研究旨在通过优化供体细胞和受体卵母细胞的制备方法来改进猪体细胞核移植（SCNT）技术。成人和胎儿成纤维细胞，卵丘和输卵管细胞被用作供体细胞，并在体内和体外成熟的卵母细胞被用作受体卵母细胞。监测融合和发育至囊胚期的百分比、囊胚与2-细胞胚胎的比率以及囊胚的细胞数作为实验参数。实验1将四种不同类型的供体细胞分别移植到体外成熟的去核卵母细胞中，胚胎成纤维细胞核移植获得的囊胚数显著高于其他细胞核移植获得的囊胚数（P < 0.05）。(15.9%与3.1-7.9%）。对于使用胎儿成纤维细胞的SCNT，将继代培养物的数量增加至15倍并没有提高发育至囊胚阶段的能力（12.2-16.7%）。在实验2中，将胎儿成纤维细胞转移到体内或体外成熟的去核卵母细胞中。当两种类型的卵母细胞进行孤雌激活作为一个初步的控制处理，一个显着增加囊胚形成被发现在体内成熟与体外成熟的卵母细胞相比（36.4%对29.5%）。然而，没有改善SCNT使用在体内成熟的卵母细胞。总之，供体体细胞的类型对猪SCNT后的发育是重要的，胎儿成纤维细胞是最有效的检查细胞。以传代培养1-10次的胎儿成纤维细胞为供体核质，以体内外成熟的卵母细胞为受体核质，建立了一个重复性好的体外培养体系。(C)2002年爱思唯尔科技有限公司All rights reserved.
This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells.(15.9% versus. 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast. (C) 2002 Elsevier Science Inc. All rights reserved.