Application Of Haploid Embryonic Stem Cells In Research On Early Embryonic Development

Early embryo development is a fine and rigorous process,which relates to the changes of histone modification,the interaction between the male and female genomes,the regulation and change of methylation,etc.in the early embryonic development.It is of great significance to study the mechanism of early embryonic development in developmental biology,reproductive biology and regenerative medicine,therefore,early embryonic development has been the focus of research.It is difficult for people to study the development of early embryos for lacking of a good research tool,due to the specificity of early embryo materials.Haploid embryonic stem cells(ha ESCs)is a new type of embryonic stem cells in recent years.The feature is that it contains only one set of chromosomes,and has the characteristics of normal embryonic stem cells,with the ability of unlimited proliferation in vitro and differentiation.Haploid embryonic stem cells is divided into parthenogenetic haploid embryonic stem cells and androgenetic haploid embryonic stem cells.Because of the diversity of its haploid origin,it can be used to simulate a single set of male and female genomes.At the same time,it has the characteristics of pluripotent and unlimited proliferation in vitro,which provides many favorable conditions for gene manipulation in vitro,and provides a good tool for gene function research.At the same time,the establishment of haploid embryonic stem cell line provides a new channel for the study of animal genetic modification and transformation.In the early embryo development,the interaction between male and female genome and the changes of the surface modification have been in a dynamic change,from the formation of fertilized eggs from sperm and oocyte.However,the study on the interaction of the genome and the modification of the surface is very difficult because of the difficulty of early embryo manipulation,and it is lack of effective research methods.Haploid embryonic stem cells provide a new research method for the interaction between male and female genomes.Genomic imprinting is one of the most important biological phenomena in the whole life.Imprinted gene is an important guarantee for the development of individuals.Because of the particularity of the material,it is difficult to collect the imprinted gene,and the research method of imprinting gene is single and difficult.Haploid embryonic stem cells is convenient for gene manipulation,so it is a good material for studying imprinted genes.In this study parthenogenetic embryonic stem cells were obtained by parthenogenetic activation,at the same time,we obtained the haploid embryonic stem cells by using the method of sperm cloning,then,we detected the parthenogenetic and androgenetic embryonic stem cells,we found that it had the normal haploid karyotype and the characteristics of normal embryonic stem cells.Then we used parthenogenetic and androgenetic haploid embryonic stem cells as tools to study the early embryonic development.First of all,we used the parthenogenetic haploid embryonic stem cells to carry out the cell fusion,and the tetraploid compensation experiments were conducted to study whether the interaction between the male and female genome in the early stage of blastocyst stage is necessary for the development of the animals.We successfully obtained the parthenogenetic and androgenetic haploid embryonic stem cells line.The cell line has a normal diploid karyotype,and it has the ability to differentiate into three germ layers in vitro and the diploid germ line chimeras,which proves that it has the ability of normal ES cells.Finally,we obtained the tetraploid mice by the tetraploid compensation experiment,the experiment is the gold standard for the highest level of cell viability,the experiment also proved that in the blastocyst stage,male and female genome interaction on animal development maturity is not required.At the same time,it is also proved that the interaction between male and female genomes is not necessary for the development of animals before the blastocyst stage.Secondly,we found that the DNA methylation modification of parthenogenetic haploid embryonic stem cells has the characteristics of decreasing with the increasing of cell generation,especially such phenomena located in the imprinting control region,at the same time,Intracytoplasmic sperm injection parthenogenetic haploid embryonic stem cells mice can only develop to E13.5 days due to abnormal imprinting and other reasons.These characteristics of parthenogenetic haploid embryonic stem cells provide a good tool for studying the function of imprinted genes,we modified the parthenogenetic haploid embryonic stem cells in different regions to mark,mark effect on early embryonic development.We modified the different imprinted regions of parthenogenetic haploid embryonic stem cells to have the effect of imprinting on early embryonic development.IGDMR gene silencing of parthenogenetic haploid embryonic stem cells was obtained by homologous targeting of DMR imprinting region of IGDMR The cells support the development of the animals,but affect the survival of the cells.Then,based on the modified of IGDMR region,we have homologous recombination in the H19 area,through knockdown the-4kb to +1kb and-10 kb to +4kb area of H19 transcription start site upstream,we found that In the haploid embryonic stem cell cytoplasm after injection having the IGDMR/ 5kbh19 delta knockout could not be obtained the survival mice,but not only did the animals have a higher rate of maturity,but the animals survived to adulthood in the haploid embryonic stem cell cytoplasm after injection having the IGDMR/ 5kbh19 delta knockout.But its weight and behavior are still abnormal compared with the wild type.Finally,we through the IGDMR/?15kb H19 knockout haploid embryonic stem cells on the basis of the overexpression of IGF2 was microinjected into the cytoplasm of the formation of IGDMR/?15kb H19/IGF2 cell line,the maturity of animal behavioral movement ability of open field test has improved,but not completely improved.In this study,we have constructed a new type of novel cell,which is used to study the interaction and imprinting regulation in the early embryonic development.It not only answered some difficult problems in early embryo development,but also provided a new idea for the study of early embryo development.Haploid embryonic stem cells plays an important role in the preparation of mitochondrial models,the interaction of different genomes and the reproductive biology.