A New Approach to Generate Transgenic Animals

产生转基因动物的新方法

• 批准号: 6669588
• 负责人: MARTIN DYM
• 金额: $ 23.28万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6669588
• 关键词: Sertoli cells; cell differentiation; cell proliferation; fertility; functional /structural genomics; gene expression; gene mutation; genetic screening; genetically modified animals; in situ hybridization; laboratory mouse; lac operon; morphology; nucleic acid purification; southern blotting; sperm; spermatogenesis; stem cell transplantation; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Spermatogenesis occurs in the testis and it is the process by which the male germ line stem cells (type A spermatogonia) divide and differentiate to produce sperm. These stem cells can be considered as a kind of "immortal" germ cell because they are present from birth to death and they have the capacity to give rise to new stem cells and as well to sperm that pass genetic material to the next generation. Thus, they represent a very valuable resource for experimental modification of the mammalian genome. Recently, LacZ transgenic mice have been created by using the technology referred to as male germ line stem cell transplantation. Classical approaches for producing transgenic animals using embryonic stem (ES) cells require labor intensive, time-consuming, and expensive procedures. Germ line stem cell transplantation may markedly simplify the classical strategies and could be an alternative technique for mutagenesis in order to identify the function of genes in the sequenced genome. However, the current limitation of this transplantation method is low efficiency and random insertion of exogenous DNA into the host genome since it is very difficult to transfer DNA into primary cultures of type A spermatogonial stem cells and impossible to select them in vitro. We recently reported the development of an immortalized male germ line stem cell, a type A spermatogonial stem cell line. This cell line can be easily manipulated in vitro to introduce exogenous DNA and to target sequence-specific sites in the genome. Importantly, the cell line can also be induced by stem cell factor to differentiate into spermatocytes and spermatids in vitro. Thus, it has very high potential to produce mature functional sperm after transplantation into recipient sterile seminiferous tubules and then the transfer of a modified genome to offspring. The aim of this proposal is to transplant our new spermatogonial cell line into sterile recipient males and then to determine whether the male recipients can father offspring. Extension of this work is to utilize our cell line to make insertional mouse mutants to facilitate the identification of gene function in the mouse genome. This research will benefit the study of human inherited disease and development as well as the study of the molecular genetics of drug addiction.

描述（由申请人提供）： 精子发生发生在睾丸中，是雄性生殖系干细胞（A 型精原细胞）分裂和分化产生精子的过程。这些干细胞可以被认为是一种“不朽”的生殖细胞，因为它们从出生到死亡都存在，并且有能力产生新的干细胞以及将遗传物质传递给下一代的精子。因此，它们代表了哺乳动物基因组实验修饰的非常有价值的资源。最近，利用雄性生殖系干细胞移植技术培育出了 LacZ 转基因小鼠。 使用胚胎干 (ES) 细胞生产转基因动物的经典方法需要大量劳动力、耗时且昂贵的程序。生殖系干细胞移植可以显着简化经典策略，并且可以成为诱变的替代技术，以鉴定测序基因组中基因的功能。然而，目前这种移植方法的局限性是效率低且外源DNA随机插入宿主基因组，因为将DNA转移到A型精原干细胞的原代培养物中非常困难，并且不可能在体外对其进行选择。 我们最近报道了永生化雄性生殖系干细胞（A 型精原干细胞系）的开发。该细胞系可以在体外轻松操作，以引入外源 DNA 并靶向基因组中的序列特异性位点。重要的是，该细胞系还可以在体外被干细胞因子诱导分化为精母细胞和精子细胞。因此，在移植到受体不育的生精小管中，然后将修饰的基因组转移给后代后，它具有非常高的潜力产生成熟的功能性精子。该提案的目的是将我们的新精原细胞系移植到不育的雄性受体体内，然后确定雄性受体是否能够生育后代。这项工作的延伸是利用我们的细胞系制造插入小鼠突变体，以促进小鼠基因组中基因功能的鉴定。这项研究将有利于人类遗传疾病和发育的研究以及药物成瘾的分子遗传学研究。