Transgenic Goats Produced by Germ Cell Transplantation

描述(申请人提供)：这项建议的目标是通过雄性生殖细胞转基因和移植来操纵雄性生殖系来产生转基因山羊。这些山羊将在乳腺特异启动子的控制下携带人类生长激素(RhGH)基因，因此转基因雌性山羊的乳汁将成为纯化人类生长激素的有效蛋白质来源。转基因山羊乳是一种安全、丰富、经济高效的重组人生物药物生产来源。GTC BioTreateutics(前身为Genzyme Transgenics Corp.)有近24个商业转基因项目正在开发中。然而，即使是最先进的方法产生转基因山羊(第一代)仍然相对低效和昂贵。在啮齿动物身上，已经开发出将转基因雄性生殖细胞移植到受体雄性体内并与这些动物交配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导入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基因组成的后代的可行性。现在，我们建议：1)在转基因生殖细胞移植后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具有商业意义的蛋白质的活转基因后代；3)建立山羊生殖细胞培养系统，使该方法普遍适用于更广泛的转基因构建。成功地通过雄性生殖系导入转基因将提高产生创始动物的效率并降低成本，这是转基因生产过程中关键的早期步骤。

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.