| It has been deminstrated that germ cells can be derived frome embryonic stem cells for its pluripotency. DAZL(Deleted in Azoospermia-Like, DAZL) is expressed early in the fetal gonads and throughout gametogenesis, it is the licensing of germ cell development and differentiation. But the mechanism by which DAZL regulates germ cell differention and meiosis is unclear, and so as its expression on several genes.In this study, DAZL gene is cloned, and its eukaryotic recombinant expression vectors were constructed. Then the expression of DAZL gene in vitro was ahieved and subcellular localization of the expression product was analyzed by fluorescence microscopy. Feeder-free culture system of chicken embryonic stem cells was established and overexpressed DAZL to induce cESC differentiate into germ cells in vitro. Major contents in this study are as follows:1. Cloning and sequence analysis of DAZL. We used Nest PCR to clone DAZL gene CDS, and inserted it to pMD19-T vector. Restriction enzyme digestion and sequencing confirmed that the recombinant vector pMD19-T-DAZL was correctly constructed, Sequence alignment showed its homology with DAZL cDNA of chicken from GenBank is99%, and amino acids encoded by it was exactly the same. This indicate that DAZL gene cloned was correct and can be used for following study.2. Construction of eukaryotic recombinant expression vectors and study on subcellular localization of its expression product in vitro. The eukaryotic expression vector DAZL-pEGFP-N1and pcDNA-DAZL were constructed, and transfected into CEF cells, using LipofectaminTM LTX,48h later, the Dazl-eGFP were observed under fluorescence inverted microscope. The mRNA and protein expression in vitro was detected, using RT-PCR and Western-blot methods, and using IFA to identify DAZL protein. The expression product was detected by RT-PCR and Western Blot. The results showed the Dazl-eGFP fusion protein located at the nucleus, indicating that The eukaryotic recombinant expression vectors were successfully constructed, with expression ability.3. The establishment of feeder-free culture system of chicken embryonic stem cells. The cES cells were isolated from blastoderm of the stage X embryos of chicken, and cultured with exogenous cytokines. Passaged cES clones by mouth sucker, and digest with collagenase IV combined with trypsin. Detected alkaline phosphatase activity and the corresponding antigen expression to identify its activity. The result showed that cES cells can stably passaged to6th generation culture in vitro using feeder-free culture system, expressing strong alkaline phosphatase (AKP) activity and stage-specific embryonic antigen-1indicating the maintenance in the state of undifferentiated, indicating that it can be used for following inducing experiments.4. DAZL induced chicken embryonic stem cells differentiated into germ cells. Adjusted plasmid, and proportion of palsimid and liposomes to obtain a best transfection system. Then the recombinant plasmid pcDNA-DAZL was transfected into the CEF using liposomes-mediated method, and the transfected cells were selected by G418to obtain positive cell lines. After IFA detecting the expression of DAZL protein, change the medium to induce into germ cells. Use real-time quantitative PCR (QRT-PCR) to detect the amount of gene expression, and IFA to detect the cells that derived from ESC. The results showed that use900ng plasmid and double liposomes can obtain the best transfect efficiency; over-expression of the the DAZL gene led to suppression of Nanog, but induced germ cell specific genes including Stra8,c-kit, integrina6and integrinβ1; IFA tests showed that the cells derived from ESC were positive for c-kit, integrina6, integrinβ1positive. Indicting that the cells derived from ESC were germ-like cells. |
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