Comparative evaluation of germ cell-specific promoters identifies robust drivers of transgene expression during mouse spermatogenesis.

backgroundCells or tissue-specific regulation of gene expression is a key approach for understanding gene function; hence, a robust cell- or tissue-specific promoter element is a prerequisite for any functional genomics study. The specificity and robustness of gene expression depend on transcription factors binding to the promoter region. To date, there is no comparative study to establish robust promoters for various stages of mammalian germ cell (GC)-specific gene expression. METHODS AND

resultsIn this study, the promoter regions of different testicular germ cell-specific genes like Ldhc, Tex13c, and Dazl were isolated from the mouse genome and characterized for their ability to drive germ cell-specific expression. The expression of the marker protein driven by those promoters was validated in vivo via germ cell transfection using testicular electroporation in mice. A comparative analysis of the activity of those germ cell-specific promoters was conducted, and the Ldhc promoter exhibited the strongest GC-specific expression followed by the Dazl promoter. As a proof of concept for functional application, shRNA targeting the c-Kit gene was expressed under the Ldhc promoter, thereby downregulating c-Kit expression in the mouse testis.

conclusionThese findings suggest that the Ldhc and Dazl promoters used in this study are robust and are expressed in testicular germ cells. These promoters can be used to study gene regulation during spermatogenesis to understand testicular physiology and male fertility. Moreover, these promoters can be used to express various genome-editing tools, specifically in the germ cells, with widespread application.