PRODUCTION OF PHARMACEUTICAL PROTEINS IN EGGS

这项研究的目的是建立鸡胚胎干细胞培养，目的是开发在鸡蛋中产生药物蛋白的转基因鸡。目前，嵌合体可以通过将新鲜分离的X期胚胎注射到受体X期胚胎中来获得。可以从X期胚盘细胞建立长期培养物，但从这些长期培养物中获得嵌合体是困难的。在X阶段，胚盘细胞是异质的，胚胎的某些区域比其他区域具有更多的多能细胞。据推测，来自早期胚胎（V-VII期）的细胞分化程度较低，这使它们成为胚胎干细胞系衍生的更好候选者。将用精氨酸加压素处理母鸡，以诱导V-VII期的过早产卵。将评价不同培养条件维持胚盘细胞处于未分化状态的潜力。对AP、SSEA-1和EMA-1呈阳性的培养物，将在体外产生外胚层、中胚层和内胚层衍生物，并且端粒酶呈阳性的培养物将被测试其促进体细胞嵌合的潜力。在第2阶段，将评价对生殖系的贡献。拟议的商业应用：该技术可以通过多种方式应用于蛋白质生产。首先，多能细胞可以被工程化以在输卵管和肝脏中产生蛋白质;这些蛋白质将分别沉积在蛋清和蛋黄中。其次，蛋白质可以由嵌合体产生，嵌合体包含基因改变的细胞和正常细胞，以及来自转基因创始人的股票。第三，人多克隆免疫球蛋白可以通过鸡B细胞以生殖系构型转录人DNA以产生抗体多样性来产生。

The aim of this research is to establish chicken embryonic stem cell cultures with the goal of developing genetically modified chickens that produce pharmaceutical proteins in their eggs. Currently chimeras can be obtained by injecting freshly isolated stage X embryos into a recipient stage X embryo. Long term cultures can be established from stage X blastodermal cells but it has been difficult to obtain chimeras from these longer term cultures. At stage X, the blastodermal cells are heterogeneous with certain regions of the embryo having more pluripotent cells than others. It is postulated that cells from earlier embryos (stage V-VII) are less differentiated which makes them better candidates for the derivation of an embryonic stem cell line. Hens will be treated with arginine vasopressin to induce premature ovipostion at stage V-VII. Different culture conditions will be evaluated for their potential to maintain the blastodermal cells in an undifferentiated state. Cultures which are positive for AP, SSEA-1 and EMA-1, will produce ectodermal, mesodermal and endodermal derivatives in vitro and are telomerase positive will be tested for their potential to contribute to somatic chimerism. In phase 2 contributions to the germline will be evaluated. PROPOSED COMMERCIAL APPLICATIONS: The technology can be applied to protein production in several ways. Firstly, pluripotential cells can be engineered to produce protein in the oviduct and liver; these proteins will be deposited in the albumen and the yolk, respectively. Secondly, the proteins can be produced by chimeras, which contain both genetically altered and normal cells, and in stocks derived from transgenic founders. Thirdly, human polyclonal immunoglobulins can be produced by chicken B cells by transcribing human DNA in germline configuration to generate antibody diversity.