Hofmann Andreas, Kessler Barbara, Ewerling Sonja, Weppert Myriam, Vogg Barbara, Ludwig Harald, Stojkovic Miodrag, Boelhauve Marc, Brem Gottfried, Wolf Eckhard, Pfeifer Alexander
Department of Pharmacy, Institute for Pharmacology, Center for Drug Research, Butenandtstrasse 5 (C), Ludwig-Maximilians University, 81377 Munich, Germany.
EMBO Rep. 2003 Nov;4(11):1054-60. doi: 10.1038/sj.embor.embor7400007. Epub 2003 Oct 17.
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 longstanding 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 germ-line. 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.
DNA显微注射是目前生成转基因动物最为广泛使用的方法，但通过这种方式在高等哺乳动物中实现的转基因效率极低。为解决这一长期存在的问题，我们使用携带普遍活跃启动子（磷酸甘油酸激酶，LV-PGK）的慢病毒载体将转基因导入猪胚胎。在出生的46头仔猪中，32头（70%）携带转基因DNA，其中30头（94%）表达了转基因（绿色荧光蛋白，GFP）。直接荧光成像和免疫组织化学显示，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，转基因通过种系传递。通过用携带人角蛋白K14启动子（LV-K14）的慢病毒载体感染猪胚胎，实现了组织特异性转基因表达。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分别在体外受精前后感染牛卵母细胞，导致45%和92%的感染胚胎中出现转基因表达。
