A New Approach to Generate Transgenic Animals

描述(由申请人提供)： 精子发生在睾丸，是男性生殖系干细胞(A型精原细胞)分裂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，它们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，它们是哺乳动物基因组实验修饰的非常有价值的资源。最近，利用被称为雄性生殖系干细胞移植的技术创造了LacZ转基因小鼠。使用胚胎干细胞生产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以显着简化经典的策略，并可能成为一种替代的诱变技术，以鉴定测序基因组中的基因功能。然而，目前这种移植方法的局限性是外源DNA随机插入宿主基因组，因为很难将DNA转移到A型精原干细胞的原代培养中，而且不可能在体外进行选择。我们最近报道了一种永生化的雄性生殖系干细胞，一种A型精原干细胞系。这种细胞系在体外可以很容易地被操纵，以导入外源DNA并靶向基因组中的序列特定位置。重要的是，干细胞因子也可以在体外诱导该细胞系分化为精母细胞和精子细胞。因此，它有很高的潜力在移植到受体不育的生精小管后产生成熟的有功能的精子，然后将修改后的基因组转移给后代。这项提议的目的是将我们新的精原细胞系移植到不育的接受者男性体内，然后确定男性接受者是否可以为后代做父亲。这项工作的延伸是利用我们的细胞系制造插入型小鼠突变体，以便于鉴定小鼠基因组中的基因功能。这一研究将有助于人类遗传性疾病和发育的研究以及药物成瘾的分子遗传学研究。

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.