A New Approach to Generate Transgenic Animals

产生转基因动物的新方法

• 批准号: 6727631
• 负责人: MARTIN DYM
• 金额: $ 15.52万
• 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 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727631
• 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，这是雄性生殖系干细胞（A型精子）分裂并分化以产生精子的过程。这些干细胞可以被视为一种“不朽的”生殖细胞，因为它们从出生到死亡都存在，并且有能力引起新的干细胞，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性修饰的非常宝贵的资源。最近，通过使用称为男性生殖系干细胞移植的技术创建了LACZ转基因小鼠。 使用胚胎茎（ES）细胞产生转基因动物的经典方法需要劳动密集型，耗时和昂贵的程序。生殖系干细胞移植可能明显简化了经典策略，并且可能是诱变的替代技术，以识别测序基因组中基因的功能。然而，这种移植方法的当前局限性是效率低，并且将外源性DNA随机插入宿主基因组中，因为很难将DNA转移到A型精子干细胞的主要培养物中，并且不可能在体外选择它们。 我们最近报道了一种永生的男性生殖系干细胞的发展，即A型精子干细胞系。该细胞系在体外很容易被操纵，以引入外源性DNA并靶向基因组中的序列特异性位点。重要的是，在体外，干细胞因子也可以通过干细胞因子诱导细胞系分化为精子细胞和精子。因此，在移植到受体无菌小管中，然后将修饰的基因组转移到后代后，产生成熟的功能精子具有很高的潜力。该提案的目的是将我们的新精子细胞系移植到无菌接受者男性中，然后确定男性接受者是否可以父亲后代。这项工作的扩展是利用我们的细胞系来使插入小鼠突变体促进小鼠基因组中基因功能的鉴定。这项研究将使人类遗传性疾病和发育的研究以及对药物成瘾的分子遗传学的研究。