Cloning Of Goat Zfy Gene CDS Region And Construction Of Zfy Gene Knockout Vector

The Zfy gene,also known as the Y chromosome linked zinc-finger protein transcriptional factor,is a sex-control-related gene located on the short arm of the Y chromosome(Yp),and its gene family also includes Zfx gene on the X chromosome and Zfa gene on the autosome.In this study,CRISPR/Cas9 technology was used to target the Zfy gene of Xinong Saanen Dairy goat,which laid a foundation for further study of the function of Zfy gene and the development of new sex-controlled goats.The main research results are as follows:1.Sequence cloning of goat Zfy gene CDS regionBased on the m RNA sequence information of goat Zfy gene predicted in NCBI,we designed three pairs of specific primers,and amplified the complete CDS region of the Zfy gene of Xinong Saanen Dairy goat.The 2406 bp Xiong Saanen Dairy goat Zfy gene was successfully cloned.The full-length sequence of the CDS region was aligned by TA clone sequencing and consistent with the predicted goat Zfy gene sequence on NCBI.2.Bioinformatics analysis of goat Zfy geneBioinformatics software was used to predict the full-length sequence of the cloned goat Zfy gene CDS region.It can be found that Zfy gene encodes 801 amino acids with a molecular mass of 90.37 ku.Zfy protein is an acidic protein with high hydrophilicity and structural instability;it has 66 possible protein phosphorylation sites;its protein does not have a signal peptide,so it is a nonsecreted protein.Through the homology analysis of some common species Zfy gene,the homology of Zfy gene of goat,cattle and Tibetan antelope is very high,reaching over 98%.3.Construction of goat Zfy gene targeting vector and detection of cutting efficiencyFirstly,the fetal fibroblasts of Xinong Saanen Dairy goat were isolated by tissue block adherence method,and three fetal fibroblasts with strong growth and good morphology were successfully established.Two of them were male and one was female.The screening experiment showed that the optimal concentration of puromycin was 1.0μg/m L.The titer of plasmid concentration showed that the optimal transfection amount of the targeting vector plasmid was 10μg for subsequent experiments;According to the CDS region of goat Zfy gene,8 of the highest ranked CRISPR/Cas9 potential target sites were screened by software,and then the off-target software prediction and statistical analysis were applied.Five candidate target sites with less potential miss sites were obtained by preliminary screening;Five different Cas9 targeting vectors were constructed for the five candidate loci.The T7E1 experiment was used to verify the cutting efficiency of the corresponding target sites in goat fetal fibroblasts.Two target sites with high cutting efficiency were identified for target 2 and target 4.4.Establishment of goat Zfy knockout cell lineUsing lipofection transfection,85 male monoclonal cell lines were cultured in this study,among which 46 lines were transfected with sg RNA2-PX459 vector,11 lines were mutated,the mutation rate was 23.91%;39 lines transfected with sg RNA4-PX459 vector,8 lines were mutated,the mutation rate was 20.51%;and there was no off-target effect at its potential off-target sites.It is clear that the No.2 target has better target-targeting efficiency at the cellular level.Thus,Zfy knockout cell lines which can be used for nuclear transfer is obtained.5.Identification of co-injected sg RNA and Cas9 m RNA goat embryosUsing prokaryotic injection technology,this study co-injected sg RNA2 and Cas9 m RNA into 96 goat pronuclear embryos.After sequencing,it was found that 27 embryos had fixed-point gene editing,the overall mutation efficiency reached 28.13%.And there is no off-target effect at its potential off-target site,which lays a technical foundation for the production of Zfy knockout goats by prokaryotic injection technology.