Inducing Polyploidization And Pluripotency Of Fish Cells In Vitro

Polyploidization phenomenon exists in nature,and it especially widely exists in plants,it has the vital significance in the process of formation and evolution of new species.When compared with the diploid,the polyploid has obvious advantages,such as rapid growth,infertility,strong resistance etc.In recent years polyploidization has been widely applied in agriculture,forestry,fishery and animal husbandry and so on.So the research of polyploidization is not only beneficial to the phenomenon of polyploidization in the theory of biological evolution,but also it is also have important guiding significance for polyploid breeding of animals and plants.Although using different ways of inducing polyploidy has succeeded in plants,but it is relatively less to obtain polyploid by inducing polyploidization animals in the animal.Especially in fish,Cells by in vitro inducing the polyploidy or getting stability antotetraploid somatic cells by flow cytometry sorting has not been reported.Pluripotent stem cells play an important role in researching the key issues in developmental biology.This paper combining cell culture technique,polyploid induction technology,somatic cell nuclear transfer technology and iPS induction technology,we conducted by SP600125 induced polyploidization crucian carp cells to obtain stable cell line which has the function of development of tetraploid crucian carp and induced zebrafish fibroblast cells into induced pluripotent stem cells(iPS)of related research by iPS technology.Specifically,this paper is mainly about several aspects of research.1.SP600125 induced crucian carp cell polyploidization and built a cell lineUsing the diploid carp tail fin as material,using cell culture technology,through the establishment and optimization of SP600125 in vitro induction of polyploidization method and flow cytometry sorting system,we successfully obtained a steady tetraploid crucian carp cell line.To obtain tetraploid crucian carp cell lines in the process of treating by many times of flow cytometry analysis and karyotype identification it is tetraploid crucian carp cell line.2.The development potential research of the stability tetraploid crucian carp cells inducedUsing somatic cell nuclear transfer technology,we established the tetraploid crucian carp cell line as the donor of somatic cell nuclear,and the diploid carp unfertilized egg as a receptor for nuclear transplantation.We conducted 6 transplantations,operating 922 unfertilized eggs of crucian carp,of which 420 eggs developed to the blastocyst,73 to the end of the gastrulation stage,and one developed to a tail abnormalities fish.In the control group(without nuclear transplantation carp eggs),922 none developed into the blastocyst stage.By flow cytometry analysis the DNA content of the reconstructed embryos of nuclear transplantation,the results show that they are tetraploid embryos.3.Using the technology of somatic cell reprogramming to reprogram zebrafish fibroblast cells into induced pluripotent stem cells(iPS)First of all,we established a stable zebrafish embryos cell lines and fin fibroblast cell lines in vitro.Then through the way of virus infection to the zebrafish embryo and fin fibroblast cell lines by importing these four pluripotency genes oct4,sox2,klf4,and c-myc factors to establish a stable iPS cell lines.The iPS cells after induction from zebrafish embryo and fin fibroblast cell lines detected alkaline phosphatase activity,the result was positive.Cell immunofluorescence identification showed that the zebrafish embryo iPS cells express OCT4,SSEA1,NANOG SOX2;but induction of zebrafish fin fibroblasts iPS cells only expressed OCT4;Zebrafish iPS cells obtained from embryos fibroblasts after infection,it can differentiate into a similar early three cellular or germ layers,ectoderm,endoderm and mesoderm,it looked like three layer structure of the embryo of the embryoid bodies(the formation of embryoid bodies is the study of stem cells in vitro differentiation ability index),and RT-PCR appraisal analysis shows that it has split into three layer source of potential of various kinds of cells.The appraisal result directly obtain zebrafish iPS cells are pluripotent.To sum up,in this article,using a small molecular compound SP600125 in vitro induced diploid carp cells polyploidization,further obtained stable through flow cytometry separation and purification of tetraploid crucian carp cell line;We found SP600125 induced polyploidization process without the Jnk signaling pathway,and p53 signaling pathway may be involved in the induction process;With tetraploid crucian carp cell line SP600125 induction of somatic cell nuclei as a donor,diploid carp unfertilized eggs as receptors,obtained tetraploid through nuclear transfer embryos.These results suggest that by combining a somatic cell induced polyploidization and somatic cell nuclear transfer technology is an effective way of producing polyploid,This research through the establishment of stable zebrafish embryos in vitro and caudal fin into fin fibroblast cell lines,in the form of virus infection to the zebrafish embryos fibroblast cell lines and fin fibroblast cell lines by importing foreign genes(oct4,sox2,klf4,and c-myc),we established a stable fish iPS cell lines,it laid a solid foundation for somatic cell reprogramming assisted breeding technology research and development,but also it provided a very useful the fish cell line tool and research platform for the developmental biology research.