PRODUCTION OF PHARMACEUTICAL PROTEINS IN EGGS

这项研究的目的是建立鸡胚胎干细胞培养，目标是开发在鸡蛋中产生药用蛋白的转基因鸡。目前，可以通过将新鲜分离的X期胚胎注入受体X期胚胎来获得嵌合体。可以从X期胚层细胞建立长期培养，但从这些长期培养中很难获得嵌合体。在X期，胚层细胞是异质性的，胚胎的某些区域比其他区域具有更多的多能细胞。据推测，早期胚胎(V-VII期)的细胞分化较少，这使得它们更适合于胚胎干细胞系的派生。在V-VII期，用精氨酸加压素诱导母鸡提前产卵。将对不同的培养条件进行评估，以确定它们维持胚层细胞处于未分化状态的潜力。AP、SSEA-1和EMA-1阳性的培养物，在体外将产生外胚层、中胚层和内胚层的衍生物，并且端粒酶阳性，将测试它们对体细胞嵌合体的贡献。在第二阶段，将评估对生殖系的贡献。拟议的商业应用：这项技术可以通过几种方式应用于蛋白质生产。首先，多潜能细胞可以被改造成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白和蛋黄中。其次，这些蛋白质可以通过嵌合体产生，嵌合体既包含基因改变的细胞，也包含正常细胞，以及来自转基因创始人的种群。第三，鸡B细胞可以通过以种系形式转录人类DNA来产生抗体多样性，从而产生人的多克隆免疫球蛋白。

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.