Genetic modification of primordial germ cells by gene trapping, gene targeting, and φC31 integrase
Genetic modification of primordial germ cells by gene trapping, gene targeting, and φC31 integrase
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DOI：
10.1002/mrd.20859
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发表时间：
2008-07-01
影响因子：
2.5
通讯作者：
Etches, Robert J.
中科院分区：
文献类型：
--
作者：
Leighton, Philip A.;Van De Lavoir, Marie-Cecile;Etches, Robert J.
生殖系定向细胞的基因组被认为受到转录沉默机制的保护，对使用培养的生殖系细胞的转基因形成障碍。我们发现，选择转基因整合到原始生殖细胞基因组需要转基因两侧的鸡P-珠蛋白绝缘子。然而，整合频率较低，插入位点的测序显示，转基因优先插入到活性启动子区域，这意味着沉默禁止恢复在其他区域的插入。当使用phi C31整合酶将转基因插入内源性假attP位点时，实现了高得多的整合频率。尽管有证据表明PGCs中存在转录沉默，但也实现了非表达基因的基因靶向。在原始生殖细胞中进行遗传修饰的能力为研究原始生殖细胞的生物学以及生产用于生物技术和发育生物学的转基因鸡提供了前所未有的机会。
The genome of germline committed cells is thought to be protected by mechanisms of transcriptional silencing, posing a barrier to transgenesis using cultured germline cells. We found that selection for transgene integration into the primordial germ cell genome required that the transgenes be flanked by the chicken P-globin insulator. However, integration frequency was low, and sequencing of the insertion sites revealed that the transgenes preferentially inserted into active promoter regions, implying that silencing prohibited recovery of insertions in other regions. Much higher frequencies of integration were achieved when the phi C31 integrase was used to insert transgenes into endogenous pseudo attP sites. Despite the evidence for transcriptional silencing in PGCs, gene targeting of a nonexpressed gene was also achieved. The ability to make genetic modifications in PGCs provides unprecedented opportunities to study the biology of PGCs, as well as produce transgenic chickens for applications in biotechnology and developmental biology.