猪克隆胚胎激活方法优化与转人溶菌酶基因克隆猪的生产
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华南农业大学动物科学学院,河北医科大学第一附属医院,中国农业大学,中国农业大学,华南农业大学,中国农业大学,华南农业大学,中国农业大学

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国家转基因育种重大专项(2008ZX08006-001), 国家重点基础研究发展计划(973)(2011CB944202)和广东省自然科学基金(S2011040001123) 资助项目


Optimizing of Activation Protocols and Production of Transgenic Pigs Expressing Human Lysozyme by Somatic Cell Nuclear Transfer
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College of Animal Science, South China Agricultural University,the first hospital of Hebei medical university,China Agricultural University,China Agricultural University,South China Agricultural University,China Agricultural University,South China Agricultural University,China Agricultural University

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This work was supported by grants from The National Transgenic Breeding Program of China (2008ZX08006-001), National Basic Research Program of China (2011CB944202) and Guangdong Natural Science Foundation (S2011040001123)

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    摘要:

    为了提高转基因克隆效率和获得转人溶菌酶基因克隆猪,研究了不同电激活参数和化学辅助激活方法对猪克隆胚胎和孤雌胚胎体外发育的影响.结果发现:电场强度会显著影响克隆胚胎的融合率和体外发育能力(P < 0.05),电脉冲次数对克隆胚胎体外发育促进作用不显著(P > 0.05),而相同电激活条件下克隆胚胎和孤雌胚胎的体外发育能力变化趋势不同;电激活后再利用放线菌酮 细胞松弛素B(CHX CB)处理4 h能显著提高克隆胚胎的囊胚率(P < 0.05),而用二甲基氨基嘌呤(6-DMAP)处理没有提高克隆胚胎囊胚率 (P > 0.05),但6-DMAP或CHX CB处理均可显著提高孤雌胚胎的囊胚率(P < 0.05).上述结果表明,最佳的孤雌激活条件并不一定是克隆胚胎的最佳激活条件.本研究中猪克隆胚胎的最佳激活方法为1.6 kV/cm、100 μs、2次直流电脉冲间隔100 μs,再辅以CHX CB处理4 h.利用优化的激活条件成功获得了乳腺特异表达人溶菌酶的转基因猪,为猪转基因育种奠定了基础.

    Abstract:

    In order to improve the cloning efficiency and obtain human lysozyme (hLY) gene transgenic pigs, the present study was carried out to investigate the effects of different electric activation parameters and chemicals on in vitro development of embryos derived from parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). The results showed that the electric strength could influence the fusion rate and developmental ability of SCNT embryos (P < 0.05), and number of electric pulses had no significant effect on SCNT embryos development (P > 0.05), yet different variation tendency was found in developmental ability between PA and SCNT embryos under same activation parameters. The blastocyst rate of SCNT embryos was improved when treated with CHX CB for 4 h after electric activation (P < 0.05), whereas 6-DMAP did not (P > 0.05). On the contrary, either CHX CB or 6-DMAP treatment after electric pulses could improve the blastocyst rate of PA embryos, indicating that the best activation method for PA was not necessarily the best for SCNT. The best activation protocol of SCNT embryos in our study is two pulses of 100 μs, direct current of 1.6 kV/cm electric strength with 100 μs interval, and followed by CHX CB treatment for 4 h. With the activation protocol, the mammary gland expressed hLY transgenic pigs were generated. It could help for improving piglets survival rate in transgenic breeding.

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卫恒习,李俊,童佳,马育芳,高凤磊,李秋艳,张守全,李宁.猪克隆胚胎激活方法优化与转人溶菌酶基因克隆猪的生产[J].生物化学与生物物理进展,2013,40(1):72-79

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历史
  • 收稿日期:2012-02-17
  • 最后修改日期:2012-08-21
  • 接受日期:2012-08-28
  • 在线发布日期: 2013-02-28
  • 出版日期: 2013-01-20