Efficient transgenesis in farm animals by lentiviral vectors
Efficient transgenesis in farm animals by lentiviral vectors
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DOI：
10.1038/sj.embor.7400007
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发表时间：
2003-11-01
期刊：
影响因子：
7.7
通讯作者：
Pfeifer, A
中科院分区：
文献类型：
--
作者：
Hofmann, A;Kessler, B;Pfeifer, A
DNA显微注射是目前最普遍的转基因动物产生方法，但这种方法在高等哺乳动物中的转基因率极低。为了解决这个长期存在的问题，我们使用携带普遍活性启动子（磷酸甘油酸激酶，LV-PGK）的慢病毒载体将转基因传递到猪胚胎中。在出生的46头仔猪中，32头（70%）携带转基因DNA，其中30头（94%）表达转基因（绿色荧光蛋白，GFP）。直接荧光成像和免疫组化结果显示，GFP在LV-PGK转基因猪的所有组织中均有表达，包括生殖细胞。重要的是，转基因是通过生殖系传递的。组织特异性转基因表达通过用含有人角蛋白K14启动子（LV-K14）的慢病毒载体感染猪胚胎来实现。LV-K14转基因动物在皮肤的基底角质形成细胞中特异性表达GFP。最后，用LV-PGK体外受精后和体外受精前感染牛卵母细胞，分别导致45%和92%的感染胚胎表达转基因。
Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this long-standing problem, we used lentiviral vectors carrying a ubiquitously active promoter ( phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene ( green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germline. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.