Construction And Functional Evaluation Of Novel Transgenic Vectors Of Livestock

Transgenic technology as scientific innovation,success and get the preliminary application in many kinds of animals,but there are still a series of restricted problems on transgenic animal large-scale production and related products research and development.Especially the high cost and low efficiency of knockout,low efficiency of integration and low level of exogenous gene expression.Exogenous DNA into the host cell may be randomly inserted into the genome of any position,thus got different formations of integration,such as effective integration,silent integration,and toxic integration,its expression level is uneven.Exogenous gene efficient expression of a particular organization in animals is closely related to the regulatory elements on vectors.CRISPR/Cas9 system is widely applied to gene editing in recent years,becoming a transgenic technology the normal operation.It can design corresponding sgRNA according to different genes specific sites for editing.However,in the cell to edit multiple genes simultaneously is still difficult.In order to construct efficient new mammal genetically modified vectors,implement high efficiency of gene expression,and improve more effective gene editing tools,experiments in the following aspects were carried out:1 Preliminary study on the role of Sus scrofa MAR in regulating the transgenic expression of PiggyBac system in HEK293TIn order to analyse Sus scrofa MAR(Sus MAR)in the role of exogenous gene expression,in this study,PiggyBac(PB)as the backbone,build the MAR in the different position of three kinds reconstruction vectors,and the Bos Taurus MAR(Bos MAR)as the positive control.The reconstruction vectors and PB translocation enzyme(PBase+)or PB translocation enzyme controlled plasmid(PBase-)co-transfection into HEK293 T cells.After 16 days none antibiotics culture,Determination protein expression by the ?-gal testing kit,analyzing LacZ gene copy number by quantitative PCR(qPCR).The results show that the newly discovered Sus MAR sequence can increase ?-gal expression level in HEK293 T cells,and does not increase the LacZ gene copy number.This test laid a foundation for the therapeutic protein stability and high level expression.2 Establishment and functional verification of a quick and easy method to construct series sgRNA vectors in the CRISPR/Cas9 systemFor optimizing the AAV mediated CRISPR/Cas9 gene editing,improve the ability of its gene editing,this experiment was carried out the following work:(1)This study selects pX601 plasmid as the backbone,with smaller fragments tRNA replace U6 promoter,build reconstruction pX601(tRNA)viral vector,and insert genes sgRNA,transfection cell,through the T7 EI method and TA cloning sequencing detection effect of exogenous and endogenous genes editing,and test the relative expression of sgRNA by qPCR to determine efficiency of tRNA or U6 promoter.(2)Meanwhile,replace the CMV promoter,with smaller EF1? build reconstruction pX601(EF1?-tRNA)virus vector,through the T7 EI method and TA cloning sequencing detection gene editing,and qPCR method in detecting SaCas9 relative expression.And packaged into AAV-DJ virus,detect virus drops,and transduction efficiency of NIH3T3 cells to detect gene editing.(3)Furthermore,using the pX601(EF1?-tRNA)as the backbone,with a simple two-step cloning method to build series sgRNA vectors with the different length of spacers,and packing into AAV-DJ virus,transduction into NIH3T3 cells,detection gene editing through the T7 EI method.The results showed that:(1)In the experiment of the exogenous gene EGFP editing,found that tRNA group also detected the phenomenon of EGFP gene editing.At the same time,targeted on endogenous gene editing experiment,tRNA group also detected the phenomenon of endogenous gene editing;Detecting sgRNA transcription with qPCR method discovered tRNA group sgRNA relative expression was lower than U6 group.But the results of gene editing effect detected by TA cloning sequencing discovered tRNA group showed higher mutation efficiency than U6 group(about 2.5 folds).(2)Smaller EF1? instead of CMV promoter,also has the effect of gene editing,proved EF1? can serve as the promoter of Cas9;But the SaCas9 m RNA expression of EF1? group significantly lower than that CMV group;TA cloning sequencing test results show that the combination of EF1?-tRNA efficiency of gene mutations is 2times higher than the combination of CMV-U6.Reconstruction vectors packaging into AAV-DJ viruses,detect virus drops degrees found pX601-EGFP-m Pcsk9-sg2 restructuring vector which has larger AAV genome(about 5.6 kb)also packing a virus,approximate to pX601(EF1?-tRNA)-EGFP-m Pcsk9-sg2 titer(< 5 kb).However,the former positive cells amount of EGFP was significantly lower than the latter(P <0.05).And,for the Pcsk9 gene editing efficiency is lower than the latter.(3)Series sgRNA vectors contain different spacer length(0,10,20,40 bp)were successfully built by two-step cloning method,and the tandem sgRNA vectors could for multiple genes editing at the same time.To pack into AAV-DJ virus and found that four kinds of reconstruction vectors were successfully packaged,infection of NIH3T3 cells and found their gene editing results is consistent with the results of plasmid transfections,prove tRNA sgRNA series could be packaged into the virus,and generate multiple loci editing at the same time.These results confirmed that in the CRISPR/Cas9 system,tRNA substitution U6 as sgRNA promoter,does not affect Cas9 editing effect,but greatly shorten the length of AAV virus genome;And the combination of tRNA promoter and EF1? is more suitable for its application in the AAV systems;Furthermore,using two-step cloning method,can more quickly and efficiently build sgRNA series vectors,make an AAV virus can edit multiple gene loci,at the same time greatly shorten the time and save the cost to a great extent.This study may provide the theoretical basis for AAV-Cas9 combination applied to clinical gene therapy.