Gene Targeting in Rat Spermatogonial Stem Cells

描述(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，通过在大鼠中使用精原干细胞建立基因靶向技术来显著影响生物学和医学。大鼠代表了用于人类生理和疾病的最广泛使用的实验室规模的哺乳动物模型。然而，尽管它在科学上广受欢迎，但培育出具有靶向基因组修饰的大鼠的技术尚未建立。因此，这项应用的长期目标之一是开发一种技术，允许科学家使用精原干细胞来生产具有特定基因组修改的转基因大鼠模型；这种动物模型将在回答与健康相关的重要问题方面代表小鼠的替代方案。这项应用的第二个长期目标将是利用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，这项建议中的近期目标是通过使用Cre-lox(特定目标1)和同源重组(特定目标2)技术在特定的基因组位置对培养中的大鼠精原干细胞进行基因改造。修改后的干细胞将在培养中使用阳性和阴性可选标记进行扩增。选定的品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，然后通过自然交配用于目标大鼠等位基因的生殖线传播。由于实验室大鼠在科学上的流行，通过拟议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，从而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、药物成瘾、心血管和肾脏疾病、免疫疾病、胃肠道疾病、肺部疾病、营养、衰老、生殖和基于细胞的治疗的研究。由于实验室大鼠在科学上的流行，通过拟议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，从而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、药物成瘾、心血管和肾脏疾病、免疫疾病、胃肠道疾病、肺部疾病、营养、衰老、生殖和基于细胞的治疗的研究。

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.