Lee S H, Gupta M K, Han D W, Han S Y, Uhm S J, Kim T, Lee H T
Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, 1 Hwayang-dong, Gwangjin-Gu, Seoul, 143 701, South Korea.
Poult Sci. 2007 Oct;86(10):2221-7. doi: 10.1093/ps/86.10.2221.
Transgenic chickens, ubiquitously expressing a human protein, could be a very useful model system for studying the role of human proteins in embryonic development as well as for efficiently producing pharmaceutical drugs as bioreactors. Human parathormone (hPTH) secreted from parathyroid glands plays a significant role in calcium homeostasis and is an important therapeutic agent for the treatment of osteoporosis in humans. Here, by using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein, we generated transgenic chickens expressing hPTH under the control of a ubiquitous Rous sarcoma virus promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage. After 21 d of incubation, 42 chicks hatched from 473 retrovirus-injected eggs. All 42 living chicks were found to express the vector-encoded hPTH gene in diverse organs, as revealed by PCR and reverse transcription-PCR analysis by using primer pairs specific for hPTH. Four days after hatching, 6 chicks died and 14 chicks showed phenotypic deformities. At 18 wk of age, only 3 G(0) chickens survived. They also released the hPTH hormone in their blood and transmitted the hPTH gene to G(1) embryos. However, although the embryos were alive at d 18 of incubation, none hatched. An electrochemiluminescence immunoassay further showed that the hPTH expression level was markedly elevated in mammalian cells infected by the retrovirus vector. Thus, we demonstrated that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, not only could be an efficient bioreactor for the production of pharmaceutical drugs, but also could be useful for studies on the role of human proteins in embryonic development. To our knowledge, this is the first report on the production of a human protein (hPTH) in transgenic chickens under the control of a ubiquitous promoter by using a replication-defective Moloney murine leukemia virus-based retrovirus vector system.
普遍表达人类蛋白质的转基因鸡，可能是用于研究人类蛋白质在胚胎发育中的作用以及作为生物反应器高效生产药物的非常有用的模型系统。甲状旁腺分泌的人甲状旁腺激素（hPTH）在钙稳态中起重要作用，是治疗人类骨质疏松症的重要治疗剂。在此，我们使用包装有水泡性口炎病毒G糖蛋白的强大的基于莫洛尼鼠白血病病毒的复制缺陷型逆转录病毒载体，生成了在普遍存在的劳氏肉瘤病毒启动子控制下表达hPTH的转基因鸡。将重组逆转录病毒注射到胚盘期新鲜产下的鸡蛋的胚下腔中。孵化21天后，473枚注射了逆转录病毒的鸡蛋孵出了42只小鸡。通过使用针对hPTH的引物对进行PCR和逆转录PCR分析发现，所有42只存活的小鸡在不同器官中均表达载体编码的hPTH基因。孵化后4天，6只小鸡死亡，14只小鸡出现表型畸形。在18周龄时，只有3只G(0)代鸡存活。它们还在血液中释放了hPTH激素，并将hPTH基因传递给了G(1)代胚胎。然而，尽管胚胎在孵化第18天时还活着，但没有一只孵化出来。电化学发光免疫分析进一步表明，逆转录病毒载体感染的哺乳动物细胞中hPTH表达水平明显升高。因此，我们证明了在普遍存在的启动子控制下表达人类蛋白质的转基因鸡，不仅可以作为生产药物的高效生物反应器，而且对于研究人类蛋白质在胚胎发育中的作用也很有用。据我们所知，这是首次报道使用基于莫洛尼鼠白血病病毒的复制缺陷型逆转录病毒载体系统，在普遍存在的启动子控制下在转基因鸡中生产人类蛋白质（hPTH）。
