A New Approach to Generate Transgenic Animals

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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.