Preliminarily Analysis Of Deleted In Azoospermia-Like (DAZL) Promoter And Its Inducer Screening And Validation

DAZL (Deleted in Azoospermia-Like, DAZL) gene expressed during the early development of germ cells and the whole stage of meiosis. DAZL was also the internal activator of initiating meiosis, which played an important role in regulation of germ cells development and differentiation. Recently, many articles have reported that DAZL gene could induce embryonic stem cells (Embryonic stem cell, ESC) to differentiate into male germ cells in vitro. Although many inducers have been used, but the induction efficiency was low. Therefore, it is important to select the optimal inducers or combination of inducers, thus to increase the expression of DAZL gene that could improve the induction efficiency of ESCs differentiate to male reproductive cells in vitro. The DAZL gene promoter in chicken and mice were successfully cloned in this study. Dual luciferase reporter assay was used to determine the core regulatory region of DAZL gene promoter. The impact of DNA methylation on promoter activity was also analysed. And the induction effects of different inducers were compared to select the optimal genetic inducer. By increasing the expression of DAZL gene in vitro, this study aimed to induce male germ cells from ESCs in vitro.1.-932～-39bp fragment sequence of Suqin yellow chicken Deleted in Azoospermia-Like (DAZL) Gene5’flanking region was first cloned by PCR, and the recombined plasmids pDAZL-EGFP and pLinker-EGFP were constructed successfully. Each of the recombined plasmids was transfected into GC-1cells, thus to qualitatively analysis the DAZL gene promoter activity of-932～-39bp fragments of5’flanking region. The results showed that the fragment of-932～-39bp had the activity as DAZL gene promoter. Different fragments of5’flanking region among-932～-39bp of chicken DAZL gene promoter were cloned and recombined into the dual luciferase reporter gene vectors——pGL3/893. pGL3/608、pGL3/334and pGL3/147. The recombined plasmids were transfected into DF-1and GC-1cells. Dual-Luciferase(?) Reporter Assay System was performed to confirm the active control area of the chicken DAZL gene. The results showed that there were some regulatory elements among-382382bp fragment sequence which played an important role in chicken DAZL gene.2. Bisulfite sequencing method was used to identify the methylation status of Suqin yellow chincken DAZL gene in-932～-39bp was identified. After adding DNA methylation transferase inhibitor (5-azacytidine), Dual-Luciferase(?) Reporter Assay System was performed to detect changes of the activity of these plasmids, thus to analyze the influence of DNA methylation on the expression and activity. Then GC-1cells were treated with single or multiple factor inducers, and Dual-Luciferase(?) Reporter Assay System was performed to identify the activity of chicken Dazl gene core promoter under different inducers. The results showed that the single factor of Am80, ATRA and the multiple factor of FSH+E2enhanced the activity of DAZL gene promoter significantly.3. The effects of different inducers were verified on mice DAZL gene promoter. Referenced to the inducers screening experiment of Suqin yellow chicken DAZL gene promoter, the effects of these inducers were further validated. The results showed that Am80and ATRA had a better induced effect on the DAZL gene promoter.