Use of primordial germ cell based chicken transgene & knockout techniques to establish animal models for studies in development & infection

鸡是发育生物学、生理学和免疫学等动物科学基础研究和应用研究的优良动物模型。因此，鸡的基因组是第一个被测序的家畜基因组。基于这些信息，候选基因在不同研究领域的鉴定及其功能表征现在是可能的。在过去，由于缺乏适当的转基因技术，在鸡体内进行功能性研究是困难的。鸡原始生殖细胞（PGC）长期培养方法的最新进展为操纵其基因组、产生转基因和敲除鸟类开辟了新的途径。该项目将首先专注于从一种被称为0号线的高度近交系的鸡株中产生PGCs。该细胞系允许细胞转移和组织移植研究，而不会有移植排斥的风险。0号线PGCs将用于插入编码绿色荧光蛋白（eGFP）的DNA。将绿色荧光转基因PGCs注射到0系胚胎中，产生转基因种系的鸟类。在交配两代后，产生的后代将在所有细胞中表达eGFP。这些鸡的荧光细胞随后将用于细胞转移。该项目的第二个目标是获得基因敲除鸟，用于鸡B细胞生物学的进一步研究。使用合适的靶向载体，可以敲除B细胞受体的轻链和重链。从这些PGCs衍生的基因敲除的鸟类将不能在前体细胞上表达功能性的B细胞受体。因此，这些细胞将无法接收到适当的生存信号以进一步分化，并将在归巢到B细胞发育的中心器官法氏囊之前死亡。这项工作将导致第一个基因敲除鸟类，并为鸟类B细胞研究提供一个极好的模型。

The chicken is an excellent animal model for basic and applied research in animal sciences like developmental biology, physiology and immunology. As a consequence, the chicken genome was the first domestic animal genome to be sequenced. Based on this information, the identification of candidate genes in the different research fields and their functional characterisation is now possible. In the past, functional in vivo studies had been difficult to perform in chickens due to a lack of appropriate transgene technologies. Recent advances in long term culture methods of chicken primordial germ cells (PGC) have opened new ways to manipulate their genome and to generate transgenic and knock out birds. This project will first focus on the generation of PGCs from a highly inbred chicken strain called line 0. This line allows cell transfer and tissue transplantation studies without the risk of transplant rejection. Line 0 PGCs will be used for insertion of a DNA coding for a green fluorescent protein (eGFP). Green fluorescent transgenic PGCs will be injected into line 0 embryos to generate germ line transgenic birds. After mating for two generations the resulting offsprings will express eGFP in all cells. Fluorescent cells from these chickens will subsequently be used for cell transfer.The second aim of this project is to obtain gene knockout birds for additional research on chicken B cell biology. Using appropriate targeting vectors the light and the heavy chain of the B cell receptor will be knocked out in line 0 PGCs. Knock out birds derived from these PGCs will not be able to express a functional B cell receptor on precursor cells. As a consequence, these cells will not receive appropriate survival signals for further differentiation and will die prior to homing into the central organ for B cell development, the bursa of Fabricius. This work will lead to the first gene knock out bird published and to an excellent model for B cell research in birds.