Gene Targeting in Rat Spermatogonial Stem Cells

描述（由申请人提供）：该项目的目标是在尚未获得多能大鼠胚胎干细胞的情况下，利用精原干细胞在大鼠体内建立基因靶向技术，从而对生物学和医学产生重大影响。大鼠是人类生理学和疾病的最广泛使用的实验室规模的哺乳动物模型。然而，尽管它在科学上广泛流行，但生产具有靶向基因组修饰的大鼠的技术尚未建立。因此，这项应用的一个长期目标是开发技术，使科学家能够使用精原干细胞生产具有特定基因组修饰的转基因大鼠模型；在回答重要的健康相关问题方面，这种动物模型将成为小鼠的替代品。这项应用的第二个长期目标将是利用所产生的技术来揭示精原干细胞的分子生物学、生理学和多能性。为了实现这些长期目标，本提案的近期目标是利用Cre-lox （Specific Aim 1）和同源重组（Specific Aim 2）技术对特定基因组位点培养的大鼠精原干细胞进行遗传修饰。修改后的干细胞将在使用阳性和阴性可选择标记物的培养中扩增。选择的细胞系将被移植到大鼠睾丸中，在那里它们将发育成转基因精子，然后通过自然交配将目标大鼠等位基因用于种系传播。由于实验大鼠在科学领域的普及，通过提出的技术使用精子干细胞产生的动物模型将极大地促进许多不同医学领域的研究，对公共卫生问题产生积极影响。这将包括在大鼠身上进行的关于神经和感觉障碍、吸毒成瘾、心血管和肾脏疾病、免疫疾病、胃肠疾病、肺部疾病、营养、衰老、生殖和基于细胞的疗法的研究。由于实验大鼠在科学领域的普及，通过提出的技术使用精子干细胞产生的动物模型将极大地促进许多不同医学领域的研究，对公共卫生问题产生积极影响。这将包括在大鼠身上进行的关于神经和感觉障碍、吸毒成瘾、心血管和肾脏疾病、免疫疾病、胃肠疾病、肺部疾病、营养、衰老、生殖和基于细胞的疗法的研究。

DESCRIPTION (provided by applicant): The goal of this project is to significantly impact biology and medicine by establishing gene targeting technology in the rat using spermatogonial stem cells given pluripotent rat ES cells have not been obtained. The rat represents the most widely used laboratory-scale mammalian model for human physiology and disease. However, despite its wide popularity in science, technology to produce rats with targeted genomic modifications has yet to be established. Therefore, one long-term goal of this application is to develop technology that will allow scientists to use spermatogonial stem cells to produce transgenic rat models with specific genomic modifications; such animal models will represent alternatives to the mouse in terms of answering important health-related questions. A second long term goal of this application will be to use the generated technology to unravel the molecular biology, physiology and pluripotent nature of spermatogonial stem cells. To realize these long-term goals, the immediate goals in this proposal are to genetically modify rat spermatogonial stem cells in culture at defined genomic sites by using Cre-lox (Specific Aim 1) and homologous recombination (Specific Aim 2) technology. The modified stem cells will be expanded in culture using positive and negative selectable markers. The selected lines will be transplanted into rat testes where they will develop into transgenic spermatozoa and then used for germline transmission of the targeted rat alleles through natural mating. Due to the popularity of the laboratory rat in science, animal models generated using sperm stem cells by the proposed technology will have a positive impact on public health issues by greatly facilitating research in many different medical fields. This would include studies in the rat on neurological and sensory disorders, drug addiction, cardiovascular and renal disease, immune disease, gastrointestinal disease, pulmonary disorders, nutrition, aging, reproduction and cell based therapies. Due to the popularity of the laboratory rat in science, animal models generated using sperm stem cells by the proposed technology will have a positive impact on public health issues by greatly facilitating research in many different medical fields. This would include studies in the rat on neurological and sensory disorders, drug addiction, cardiovascular and renal disease, immune disease, gastrointestinal disease, pulmonary disorders, nutrition, aging, reproduction and cell based therapies.