The Research On Sex Identification Of Embryos By The Technology Of Marked Y Chromosome Mediated By CRISPR/Cas9

Sex control is of great significance for accelerating the breeding speed of excellent females and improving the production efficiency of limited traits animal husbandry,and sex identification of early embryo is one of the most important method for sex control.In XY sex-determined mammals,Y chromosome determines the male gender.Therefore,if Y chromosome can be labeled,it would be easy to distinguish gender between male and female individuals.The purpose of this study was to establish a rapid and non-invasive method to sex mammal's early embryos by a labeled Y chromosome and gene editing,which provides a new idea for the sex control of mammals.Based on this,the following attempts were made:firstly,this study optimized the integration efficiency of exogenous gene mediated by CRISPR/Cas9.According to that,we obtained the transgenic male mice line integrated e GFP gene in Y chromosome by CRISPR/Cas9mediated HDR,the e GFP gene can be transmitted to the genome of male offspring with the separation of the Y chromosome.Therefore,the expression of green fluorescent protein gene can intuitively indicate the sex of early embryo.To determine the usefulness of this method,we further modified the gene of Bama pig kidney fibroblasts(PKF)and buffalo fetal fibroblasts(BFF)cells using CRISPR/Cas9 mediated HDR,and successfully obtained fibroblast cell lines with an exogenous gene e GFP inserted in Y chromosome.Then,we successfully produced Y-Chr-e GFP transgenic cloned embryos by somatic cell nuclear transfer.The main works and results of this thesis are summarized as follows:1.Optimization the integration efficiency of exogenous gene mediated by CRISPR/Cas9To improve the efficiency of HDR,in this chapter,three template vectors of different lengths were constructed for targeting mouse Tubb3 and Actb gene in mouse cell lines.As a result,we found that the longer homologous arm of the template vector,the higher efficiency of homologous recombination.Then,we compared the HDR efficiency of three different donor plasmids with 300 bp,800bp and 1200 bp homologous arm in BFF cells.The results also indicated that the donor plasmid with 1200 bp homologous arm is more efficient than others.Furthermore,our research demonstrated that addition of small molecular inhibitor PFT-?of P53 can improve the HDR efficiency in BFF cells.Besides,this study also compared the targeted integration efficiency of HDR and HMEJ(homology mediated end joining),the results showed that the integration efficiency mediated by HMEJ was slightly higher than HDR in some types of cells,but there was no significant difference(P>0.05).2.Identification the sex of pre-implantation mouse embryos using a marked Y chromosome and CRISPR/Cas9In this chapter,mice models were used as the research subjects,Y-Chr-e GFP transgenic male mouse line harboring an e GFP gene in the intergenic sequence between Uty and Ddx3y gene of Y chromosome were produced by zygotes cytoplasmic injection and embryo transfer.Then,collecting the embryos by mating Y-Chr-e GFP transgenic male mice with wild type female mice,all the GFP~+embryos were male,and the GFP~-embryos were female.To further confirm the accuracy of this method,nested PCR was performed,and the results showed that the accuracy of sex identification by fluorescence was consistent with method of PCR.In addition,this research demonstrated that there was no significant difference in the the gender ratio of offspring between Y-Chr-e GFP transgenic and non-transgenic male mice.3.Generation of Y-Chr-e GFP transgenic cloned bama minipig embryos using CRISPR/Cas9 mediated HDRFirstly,kidney fibroblasts cells of Bama minipigs harboring e GFP gene in Y chromosome was produced using CRISPR/Cas9 mediated HDR,and the accuracy of target locus was confirmed.Then,we compared the growth characteristic of transgenic cells with non-transgenic PKF cells,the results showed that the growth rate and cell proliferation rate of Y-Chr-e GFP transgenic cells were significantly lower than non-transgenic fibroblast cells.Finally,Y-Chr-e GFP Bama pig transgenic cloned embryos was successfully obtained by somatic cell nuclear transfer.4.The growth,proliferation and expression level of epigenetic related genes in Y-Chr-e GFP transgenic buffalo fetal fibroblast cells and the generation of transgenic cloned embryosFirstly,buffalo fetal fibroblasts cells harboring e GFP gene in Y chromosome was generated using CRISPR/Cas9 mediated HDR and the accuracy of target locus was also confirmed.Then,the growth characteristic of Y-Chr-e GFP transgenic cells were analyzed,the results showed that the growth rate and cell proliferation rate were significantly lower in transgenic than in non-transgenic buffalo fetal fibroblast cells;furthermore,the results of real-time quantitative PCR showed that the expression levels of DNA methylation-related genes DNMT1 and DNMT3a were similar;however,the expression levels of the histone deacetylase genes HDAC1,HDAC2,and HDAC3 were significantly higher(p<0.05)in Y-Chr-e GFP transgenic BFF cells compared with non-transgenic cells;Finally,Y-Chr-e GFP transgenic BFFs were used as donor cells for SCNT,the blastocyst rates of buffalo cloned embryos were similar;however,the cleavage rates were significantly lower compared with control.In conclusion,this study successfully identified the sex of pre-implantation mouse embryos using a marked Y chromosome mediated by CRISPR/Cas9,which provides a rapid and non-invasive method for sexing mammals'early embryos.Moreover,this study also produced pig and buffalo transgenic cloned embryo harboring a marked Y chromosome,which provides new ideas and methods for sex control of large animals,meanwhile lays the foundation for the generation of genetically modified livestock by somatic cell cloning mediated gene modification.