Expression And Function Analysis Of Medaka Prdm14

Stem cell is an important subject in the field of biology research,especially the regulatory mechanisms of its pluripotency.In mouse and human,PRDM14 is known as a core transcription factor of the regulatory network.It plays a key role in the regulation of pluripotency,reacquisition of potential pluripotency,establishment of germ cells and epigenetic reprogramming.But the function of PRDM14 in other species remains to be investigated.Medaka(Oryzias latipes)is a model fish widely used in various kinds of research.In this study,we cloned medaka Prdm14 and analyzed the gene structure and evolutionary relationships;prokaryotic expressed Prdm14 protein and prepared its polyclonal antibody;analyzed the expression pattern of Prdm14 in adult tissues and embryos by using RT-PCR,Western blot and in situ hybridization(ISH);moreover,we analyzed the function of medaka Prdm14 in stem cell by over expression and in early embryos by loss of function,respectively.We aimed to provide a foundation for further study of molecular regulatory mechanisms in fish stem cell.The main results are summarized as follows:1.We cloned and characterized the CDS of medaka Prdm14,which is 1761 bp consisted of 8 exons and encoding a protein of 586 residues.We compared the Prdm14 protein amino acid sequences with some common species,the results showed that it is highly conserved in PR/SET domain and zinc fingers.Phylogenetic analysis showed that the medaka Prdm14 was grouped into Prdm14 homologues clades and clustered together with the teleost fish.These results suggested that medaka Prdm14 protein was evolutionarily conserved.2.The recombinant expression vector pET32a-Prdm14?600was constructed and transformed into Escherichia coli BL21(DE3).The recombinant protein(60 kD)was obtained after being induced with isopropyl-?-d-thiogalactoside(IPTG).Then the purified protein was used to immunize rabbit(Oryctolagus cuniculus)to generate polyclonal antibody.The titer and specificity of the antibody were detected by ELISA and Western blot.Results showed that the antibody reconginzed specifically with Prdm14 protein in medaka organs and Prdm14: EGFP fusion protein.In conclusion,we generated a polyclonal antibody with specific reconginzation of medaka Prdm14.3.The expression pattern of Prdm14 in medaka tissues were investigated by RT-PCR and Western blot.The results showed that medaka Prdm14 exhibited wide expression in many organs,with higher expression in ovary,testis and brain.The in situhybridization(ISH)on testicular and ovarian sections and whole ovary indicated that Prdm14 mRNA distributed throughout oogenesis and spermatogenesis,which suggested Prdm14 may participate in regulating the formation of germ cells.4.The expression of madaka Prdm14 during early developmental stages was also detected by RT-PCR,qRT-PCR and whole mount in situ hybridization(WISH)analysis.The results indicated that Prdm14 expressed start from blastula stage,and the expression level was highest in gastrula stage then declined in subsequent stages.5.Prdm14: EGFP expression vector was transfected into HepG2 cells and Prdm14 fusion protein was localized in the nucleus,which coincided with the characteristic of transcription factors.We overexpressed Prdm14 in SG3 and examined the expression of pluripotency-associated factors by qRT-PCR,which showed Nanog,Oct4,Sox2 and Lin28 were up-regulated.In addition,the SG3 cells overexpressed with Prdm14 proliferated faster than control cells.Taken together,medaka Prdm14 may also participate in the regulation network of pluripotency.6.The loss of function by CRISPR/Cas9 system was performed to investigate the function of Prdm14 in embryonic development.Compared with control group,large number of malformation and dead embryos were observed after the injection of CRISPR/Cas9 in the knockout group.We also validated the deletion near the gRNA target site in the genomic sequence in all mutants.Whereas injection of Prdm14 mRNA with gRNA and Cas9 mRNA reduced the death rate and malformation rate.Therefore,the deficiency of Prdm14 may result in death or malformation of medaka embryos,and Prdm14 mRNA could rescue the loss of function.