Construction Of Dazl-, Ddx4-Expression Vectors And Preliminary Study Of MiR302/367-induced Reprogramming

Stem cell is a self-renewing, clonal entity which can generate several differentiated celltypes. Stem cells are widely used in basic research, being the basis of regenerative medicineresearch as well. The ultimate goal of regenerative medicine is to replace lost or damagedcells, which can potentially be accomplished using the processes of dedifferentiation,transdifferentiation or reprogramming. Transdifferentiation refers to the conversion of cellsfrom one differentiated cell type to another, without reversion to pluripotent cells. Accordingto reported research, there are two strategies for transdifferentiation. One is direct induction ofseveral lineage-specific transcription factors or genes. The other scheme is creating anunstable state with transient overexpression of pluripotent transcription factors and theninducing differentiated cells with small molecules.Germ cells are highly specialized populations of cells which are responsible for thecontinuation and evolution of the species. Germ cell research offers promising application incell biology, developmental biology, livestock production and regenerative medicine. Thegeneration of germ cells and initiation of meiosis have always been hotspots in life scienceresearch. Some scientists devote themselves to study germ cell differentiation induced fromES cells and iPS cells. However, the tumorigenicity of pluripotent stem cells and lowdifferentiation efficiency urge scientists to explore more approaches to induce germ cells.Overexpression of Dazl (deleted in azoospermia-like) gene in embryonic stem (ES) cellscontributed to the induction of germ cells. Dazl gene is specifically expressed in mouse maleand female germ cells. Disruption of the Dazl gene leads to loss of germ cells and completeabsence of gamete production. Ddx4(DEAD (Asp-Glu-Ala-Asp) box polypeptide4, mousevasa homolog) gene is exclusively detected in germ cells. Loss of Ddx4gene in the mouseaffects differentiation of the germ cells, resulting in sterility. MiR302/367cluster is capable ofreprogramming somatic cells into induced pluripotent stem cells, with a possible higherefficiency than pluripotent transcription factors.In this study, we cloned mouse germ cell-specific genes Dazl and Ddx4, and constructedthe eukaryotic expression vectors, providing potential materials for further research ofsomatic cells transdifferentiating into germ cells. In addition, we constructed lentivirus vector yielding miR302/367. After infection, HEK293T cells were cultured in N2B27-based medium.We preliminarily researched the roles of miR302/367in dedifferentiation, reprogramming,and germ cell differentiation, providing potential materials and induction methods for furtherresearch of somatic cells transdifferentiating into germ cells.1. Construction of mouse germ cell-specific genes Dazl-and Ddx4-expressioneukaryotic vectorsAccording to GenBank database, we designed amplification primers for mouse Dazl andDdx4coding sequence (CDS) with Primer Premier5software. Mouse testis cDNA was usedas template, and RT-PCR was perfomed to amplify the products before cloned into pMD18-Tvectors. The correct clones confirmed by DNA sequensing were transferred into pEGFP-C1orpIRES2-EGFP vectors respectively to construct eukaryotic expression vectorspDazl-EGFP-C1or pDdx4-IRES2-EGFP. The recombinant plasmids were transfected intoHEK293and NIH3T3cells. The results of QRT-PCR and immunofluorescence analysisproved the overexpression of two genes and subcellular localization.2. Preliminary study of miR302/367-induced HEK293T cell reprogramming withgerm cell differentiation potentialPCR was perfomed to amplify the DNA sequence of miR302/367with mouse genomicDNA template before cloned into pMD18-T vectors. The correct clone confirmed by DNAsequensing was transferred into pCDH-CMV-MCS-EF1-Puro vector to construct lentivirusvector pCDH-miR302/367. Infected with lentivirus producing miR302/367, and cultured inDMEM/F12-and Neurobasal-based medium, with the addition of N2supplement, B27supplement, leukemia inhibitory factor (LIF), Glutamine, non-essential amino acids,β-mercaptoethanol, bovine serum albumin (BSA), PD0325901, CHIR9902, vitamin C, andbasic fibroblast growth factor (bFGF), HKE293T cells were induced to dedifferentiated andreprogrammed into iPS-like mirPS cells. RT-PCR, QRT-PCR and immunofluorescenceanalysis showed that induced mirPS cells expressed several pluripotent markers.After culturing adherent embryo bodies (EBs) for7days, the differentiated mirPS cellsexpressed markers of ectoderm, mesoderm and entoderm including chondrocytes and germcells. The mirPS cells were transplanted subcutaneously into dorsal flanks of kidney inbusulfan-treated mice, and teratoma was formed after9weeks. The immunofluorescenceanalysis showed the potential of germ cell differentiation.