| As a desirable in vitro system,fish cell lines are indispensable tools to replace some fish biological research.Isolation of species-specific cell lines were widely used in the fields of physiology,toxicology,virology,gene function,germplasm resource protection and transgenic analysis and serve as specific,basic,and low-cost study materials for specific species.The Chinese sturgeon(Acipenser sinensis)is the largest individual fish of the genus Acipenser,which lives in the southernmost latitude.The Chinese sturgeon is endangered through anthropogenic activities including over-fishing,damming,shipping,and pollution.Additionally,its biological characteristics,such as large size,slow growth and late maturity,make it difficult to recover.Several studies of Chinese sturgeon have focused on its morphology,resource ecology and reproductive biology.The establishment of Chinese sturgeon continuous cell lines are important for two broad reasons,the first at the cellular level to protect germ plasm resources of Chinese sturgeon,second,the Chinese sturgeon organization cell lines can be used as a research model in vitro,support Chinese sturgeon cell genetics and biology research.In this study,one tissue of black carp(Mylopharyngodon piceus)from fins and five tissue cell lines of Chinese sturgeon from testis,muscle,skin,liver,and fins were established,named MPF,AST,ASM,ASSK,ASL,and ASF.Up to now,MPF and all five tissue cell lines of Chinese sturgeon have been respectively subcultured over 81 and 60 passages and the suitable growth temperature of these cell lines is 28?.Besides,the cell lines were maintained in DMEM supplemented with 10% bovine serum,10 ng/m L ?-FGF,fish serum and fish embryo extract.MPF exhibited epithelial-like morphology whereas cell lines of Chinese sturgeon exhibited both fibroblast-like and epithelial-like morphology.Chromosomal analysis showed that MPF possess a modal diploid chromosome number of 48 whereas the Chinese sturgeon chromosome number is 264,which is a polyploid.The mitochondrial 18 s r RNA sequence analysis results indicated a 99% identity to the 18 s r RNA sequence of Chinese sturgeon in NCBI and a 99% identity to the 16 s r RNA sequence of black carp,further confirming the origination of these cell lines.In addition,the transfection efficiency of MPF,AST and ASM cells was tested respectively by liposome transfection,baculovirus transduction and electroporation,which demonstrated the transient transfection efficiency of MPF were respectively 8% and 24%,using liposome transfection and electroporation.Moreover,liposome transfection and baculovirus transduction are ineffective against Chinese sturgeon cells.The Chinese sturgeon cell damage is minimal and the transient transfection efficiency is 15% when using electroporation under this condition(breakdown voltage 200V;breakdown time 10 ms).Furthermore,transgenic AST and ASM cell lines were successfully established using the Sleeping Beauty Transposon system.As for MPF,the expressions of nanog and oct4 were detected in the cells,indicating that MPF might be an adult stem cell line.Besides,MPF was sensitive to Spring viremia virus(SVCV)virus and significant cytopathic effect could be observed.Meanwhile,SVCV can also proliferate strongly in MPF cells.The results above indicated that MPF cells could not only be used as an in vitro tool for the study of black carp gene function and viral pathogenic mechanism for freshwater fish research,but also a possible material for fish stem cell and associated genes research.Taken together,these newly established Chinese sturgeon cell lines could not only serve as a protection of the germplasm resources of this treasured species at the cellular level,but also become an ideal in vitro model for gene function in Chinese sturgeon after screening out the optimal transgenic methods.The development of germ cells is an important and complicated process for the growth,development and reproduction of an organism.The molecular mechanism underlying the germ cell proliferation and differentiation has always been an important subject in life sciences.Dazl,an important member of the DAZ family,is specifically expressed in germ cells of vertebrates and encoded RNA-binding protein which plays a vital role in the development and differentiation of germ cells.The black carp,the four major Chinese carp species,has become a popularly cultivated fish species in China due to its large size,rapid growth,good taste,high nutritive value and market price over other common freshwater fish,but reproductive difficulties and has longer reproductive cycle.Therefore,the identification of the marker gene daz L in black carp germ cells provides an important tool and reference for studying the development and gonadal differentiation of black carp germ cells.In this study,the full-length dazl c DNA with a total length of 2013 bp in black carp was isolated using RACE,encoding a total of 216 amino acids.Protein sequence alignment revealed this sequence contains the RRM conserved region and a DAZ repeated sequence and exhibit the highest sequence similarity of 85% with zebrafish(Danio rerio).Dazl protein was induced by constructing a recombinant expression vector and then rabbit anti-Dazl polyclonal antibody was prepared.It was verified that the Dazl antibody can specifically recognize prokaryotic expression protein,ovarian tissue endogenous protein and eukaryotic expression protein.In tissues,dazl was restricted to the gonads of black carp.Furthermore,in situ hybridization and immunofluorescence results showed that dazl transcripts expressed in early oocytes,mainly distributing in the cytoplasm stages I,II and III oocytes.During the embryogenesis,dazl firstly exhibited high expression level in early developmental stage,then gradually decreased,and finally no signal could be detected in the gastrula stage.Moreover,whole mount in situ hybridization further confirmed dazl transcripts mainly expressed in the cells of early embryos.The 1612 bp 5' flanking region of black carp dazl gene was amplified by Tail-PCR and dualluciferase plasmid cassettes of 3 different length of this sequence were constructed,termed as psi-CHECK-1612,psi-CHECK-938 and psi-CHECK-303.Subsequently,they were respectively transfected into SG3,CIK and SKOV3 cell lines to detect the core promoter region of dazl gene promoter.The results showed that-850?+88 region possess important regulatory elements for black carp dazl gene.Moreover,the reproductive cycle of black carp is long and the egg membrane of the embryo is thin,it is not suitable for gene function research,the CRISPR/Cas9 system was conducted to knock out the dazl gene in medaka(Oryzias latipes)which demonstrated that the deletion of the medaka dazl might lead to the loss of primordial germ cells(PGCs)in the medaka embryo.All the results above indicated that the black carp dazl gene is supposed to be an important factor related to the formation and development of germ cells by analyzing the expression pattern of dazl gene in black carp.Combined with the expression in other fishes and the knock out result of medaka dazl in vivo,it would provide a reference for further study of dazl gene and germ cells in black carp.In summary,first,the establishment of a rare and endangered species Chinese sturgeon cell lines,analysis of their basic biological characteristics,screening the optimal transfect method and establishment of transgenic Chinese sturgeon cell lines will contribute to the protection of the germplasm resource and provide ideal in vitro tools for the study of gene function in Chinese sturgeon.Second,the establishment of black carp fin cell line and analysis of its biological characteristics will lay the foundation for future research on gene function and virus pathogenic mechanism in black carp.Third,the dazl gene associated with germ cells in black carp was cloned and identified and confirmed its expression in the early embryonic development and in the germ cells of the gonads,further PGCs loss were observed when dazl gene was knock out in medaka,which may play a role in the formation and development of germ cells. |
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