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

鸡是动物科学如发育生物学、生理学和免疫学基础和应用研究的极好动物模型。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，在不同的研究领域中的候选基因的识别和它们的功能特性现在是可能的。过去，由于缺乏适当的转基因技术，很难在鸡中进行体内功能研究。鸡原始生殖细胞（PGC）长期培养方法的最新进展为基因组操作、转基因和基因敲除鸡的培育开辟了新的途径。该项目将首先集中于从高度近交的鸡品系（称为0号系）中产生PGCs。这条线允许细胞转移和组织移植研究，而没有移植排斥反应的风险。0号系PGC将用于插入编码绿色荧光蛋白（eGFP）的DNA。将绿色荧光转基因PGC注射到0系胚胎中以产生生殖系转基因鸟。交配两代后，所产生的后代将在所有细胞中表达eGFP。本项目的第二个目的是获得基因敲除的鸡，用于鸡B细胞生物学的进一步研究。使用适当的靶向载体，将在第0系PGC中敲除B细胞受体的轻链和重链。来源于这些PGC的敲除鸟类将不能在前体细胞上表达功能性B细胞受体。因此，这些细胞将不会接收到用于进一步分化的适当的存活信号，并且将在归巢到用于B细胞发育的中心器官（腔上囊）之前死亡。这项工作将导致第一个基因敲除鸟发表和一个很好的模型，B细胞研究的鸟类。

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.