Transgenic Goats Produced by Germ Cell Transplantation

描述（由申请人提供）：本提案的目的是通过雄性生殖细胞转染和移植操作雄性生殖系来生产转基因山羊。山羊将携带在乳腺特异性启动子控制下的人生长激素（rhGH）基因，使得转基因雌性山羊的乳汁将成为用于纯化人生长激素的有效蛋白质来源。转基因山羊奶是一种安全、丰富、成本效益高的重组人生物药物生产来源。GTC Biotherapeutics（原Genzyme Transgenics Corp.）有将近二十几个商业转基因项目正在开发中。然而，即使是用于产生转基因创始者（第一代）山羊的最先进的方法仍然相对低效和昂贵。已经在啮齿动物中开发了用于将遗传修饰的雄性生殖细胞移植到受体雄性中并使这些动物交配以产生转基因后代的方法。在该项目的第一阶段，我们成功地将生殖细胞转染和移植系统应用于山羊，并证明了受体山羊产生携带生殖细胞供体动物遗传组成的后代的可行性。现在我们建议：1）在移植转染的生殖细胞后产生产生转基因精子的山羊; 2）通过体外受精产生转基因胚胎，并产生在其乳汁中表达商业上重要的蛋白质的活的转基因后代;和3）建立山羊生殖细胞培养系统，以使该方法普遍适用于更广泛的转基因构建体。通过雄性生殖系成功引入转基因将提高效率并降低产生创始动物的成本，这是转基因生产过程中的关键早期步骤。

DESCRIPTION (provided by applicant): The objective of this proposal is to produce transgenic goats by manipulation of the male germ line through male germ cell transfection and transplantation. The goats will carry the gene for human growth hormone (rhGH) under the control of a mammary gland specific promoter such that the milk of transgenic females will be an efficient source of protein for purification of human growth hormone. The milk of transgenic goats is a safe, abundant and cost effective production source for recombinant human biopharmaceuticals. GTC Biotherapeutics (formerly Genzyme Transgenics Corp.) has nearly two dozen commercial transgenic programs in development. However, even state-of-the-art methods for generating transgenic founder (first generation) goats remain relatively inefficient and expensive. Methods have been developed in rodents for transplanting genetically modified male germ cells into recipient males and mating these animals to produce transgenic offspring. During phase I of this project we successfully adapted the systems of germ cell transfection and transplantation to goats and demonstrated feasibility that recipient goats produce offspring carrying the genetic makeup of the germ cell donor animal. Now we propose to: 1) produce goats making transgenic sperm after transplantation of transfected germ cells; 2) generate transgenic embryos by in vitro fertilization and produce live transgenic offspring carrying expressing a commercially significant protein in their milk; and 3) establish a system for caprine germ cell culture to make the approach universally applicable to a wider range of transgene constructs. Successful introduction of a transgene through the male germ line will increase the efficiency and reduce the cost of generating founder animals, which is a critical early step in the transgenic production process.