Production Of Tg(oct4:EGFP) Transgenic Zebrafish And Rapid Screening Of Homozygotes

Oct4 gene plays an important role in early embryonic development,especially in maintaining cell pluripotency.Zebrafish is one of the important model organisms,and the establishment of fluorescently labeled oct4 transgenic fish lays the foundation for stem cells research in fish.In this study,the PBLK-oct4-EGFP plasmid with the TOL2 transposon was constructed,and a green fluorescent-labeled oct4 transgenic fish(F0 generation)was obtained.Combining with gynogenetic technology,a method of quickly preparing homozygous transgenic fish is established.The main results are as follows:1.The promoter sequence of zebrafish oct4 gene was cloned.The PBLK plasmid backbone and pMD18-T-oct4 plasmid with oct4 promoter were double digest,and used the T4 ligase to ligate oct4 promoter to digested PBLK plasmid.The PBLK-oct4-EGFP plasmid with TOL2 transposon,a total length of 7338 bps,was successfully constructed.2.The mixture of PBLK-oct4-EGFP plasmid and TOL2 transposase mRNA was microinjected into zebrafish fertilized eggs,and obtained about 53% of green fluorescent embryos(F0 generation).The PCR detection showed that the egfp gene was detected in half of the F0 generation.Analysis of the expression of oct4 and egfp genes in F0 generation embryos by fluorescence quantitative PCR,there showed that the zebrafish oct4 gene had the characteristics of maternal gene expression,and the expression of oct4 gene decreases in the differentiation of embryonic cells.Observations indicated that the green fluorescence could be observed in oocytes and eggs of the transgenic zebrafish.3.Eggs of F0 transgenic zebrafish were activated by UV-irradiatedsperm of red crucian carp,and using cold shock to double the chromosomes.Then picking out the fluorescent embryos(F1 generation),Tg(oct4: EGFP)homozygous transgenic zebrafish were obtained.4.Cutting the caudal fin tissue of homozygous transgenic zebrafish,combined with in vitro cell culture technology,the Tg(oct4: EGFP)transgenic zebrafish caudal fin fibroblast cell line was established.Conclusion: oct4 gene plays an important role in maintaining cell pluripotency and it is also an important stem factor.By constructing the PBLK-oct4-EGFP plasmid,oct4 gene fluorescently labeled zebrafish was obtained.Combined with gynogenetic technique,Tg(oct4:EGFP)homozygous transgenic zebrafish were successfully prepared at the F1 generation.And at the same time Tg(oct4: EGFP)cell line was established from the homozygous transgenic zebrafish.The establishment of oct4 gene fluorescently labeled fish and fluorescently labeled cell line will be widely used in the research of developmental biology,reproductive stem cell biology,induced pluripotent stem cells,etc.Meanwhile,the new method of obtaining homozygous transgenic fish through gynogenetic and transgenic technology is helpful in the quickly preparing transgenic fish.