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转基因导入和再克隆对猪转基因克隆胚胎体外生产效率的影响。

Effect of transgene introduction and recloning on efficiency of porcine transgenic cloned embryo production in vitro.

作者信息

Uhm S J, Gupta M K, Das Z C, Kim J H, Park C, Kim T, Lee H T

机构信息

Department of Animal Biotechnology, Bio-Organ Research Center/Institute of Biomedical Science and Technology, Konkuk University, Seoul, South Korea.

出版信息

Reprod Domest Anim. 2009 Feb;44(1):106-15. doi: 10.1111/j.1439-0531.2007.01005.x. Epub 2008 Nov 18.

Abstract

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNbeta-Z or LNbeta-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro. Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNbeta-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 +/- 6.4%; 12.0 +/- 5.7%) or EGFP (57.5 +/- 6.3%; 10.1 +/- 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 +/- 8.2%; 12.3 +/- 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.

摘要

逆转录病毒介导的体细胞外源基因转染是通过体细胞核移植(SCNT)生产转基因胚胎的一种有效方法。本研究评估了使用逆转录病毒载体转染的成纤维细胞通过SCNT生产转基因胚胎的效率是否受导入的转基因影响,以及再克隆是否能进一步提高其效率。通过用LNβ-Z或LNβ-增强绿色荧光蛋白(EGFP)逆转录病毒载体转染的猪胎儿成纤维细胞进行SCNT来生产转基因克隆胚胎,并评估其体外发育能力。用LNβ-EGFP逆转录病毒载体转染的胎儿成纤维细胞通过SCNT产生的四细胞期猪胚胎的卵裂球随后被再克隆到去核的中期II卵母细胞中,并评估染色质构型变化、体外胚胎发育和基因表达。结果分析表明,使用LacZ(53.6±6.4%;12.0±5.7%)或EGFP(57.5±6.3%;10.1±4.1%)转染的成纤维细胞的猪SCNT胚胎的卵裂率和囊胚率与未转染的对照(60.9±8.2%;12.3±4.0%)没有差异(p>0.05)。卵裂球的再克隆并没有进一步提高体外发育率。有趣的是,卵裂球的核比体细胞核经历了更慢的重塑过程。克隆和再克隆胚胎均显示100%的转基因表达,且没有嵌合体的证据。总之,我们的数据表明,用复制缺陷型逆转录病毒载体转染的体细胞通过SCNT生产转基因克隆胚胎的效率不受向供体细胞中导入转基因的影响,并且四细胞期卵裂球的再克隆不能进一步提高其效率。

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