Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs
Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs
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ICSI 介导的基因转移方法在转基因猪生产中的表征
DOI：
10.1002/mrd.22015
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发表时间：
2011
影响因子：
2.5
通讯作者：
他3名
中科院分区：
文献类型：
--
作者：
Umeyama K;Saito H;Kurome M;Matsunari H;他3名
了解转基因导入卵母细胞或胚胎的行为对于评价转基因动物生产的方法是必不可少的。我们研究了通过卵胞浆内单精子注射介导的基因转移（ICSI-MGT）或原核显微注射（PN注射）转移到猪卵中的转基因的表达模式。通过ICSI-MGT引入EGFP基因产生了显著更多的具有非嵌合转基因表达的胚胎（P< 0.01）。在ICSI-MGT组中，61.5%（24/39）的胚胎在桑椹胚期的所有组成卵裂球中均呈EGFP阳性，而PN注射组中不到10%的胚胎呈EGFP阳性。使用大小从3.0到7.5 kb的三种类型的转基因，我们证实通过ICSI-MGT获得的胎儿中约有四分之一是转基因的，这表明ICSI-MGT是生产转基因猪的实用方法。通过ICSI-MGT产生的12个转基因胎儿的Southern印迹分析显示，整合的转基因拷贝数从1到300不等，转基因大小与整合拷贝数之间没有相关性。荧光原位杂交分析表明，转基因随机整合到一个单一的网站上的主机染色体。总之，这些数据表明，转基因的多拷贝、单位点整合是猪ICSI-MGT的主要结局，并且ICSI-MGT比PN注射更不可能以嵌合方式引起转基因整合。摩尔Reprod. Dev. 79：218-228，2012.© 2011 Wiley Periodicals，Inc.
Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection‐mediated gene transfer (ICSI‐MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI‐MGT yielded significantly more embryos with non‐mosaic transgene expression (P< 0.01). In the ICSI‐MGT group, 61.5% (24/39) of the embryos were EGFP‐positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP‐positive in the PN‐injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI‐MGT was transgenic, suggesting that ICSI‐MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI‐MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple‐copy, single‐site integration of a transgene is the primary outcome of ICSI‐MGT in the pig and that ICSI‐MGT is less likely than PN injection to cause transgene integration in a mosaic manner. Mol. Reprod. Dev. 79: 218–228, 2012. © 2011 Wiley Periodicals, Inc.