Construction Of Expression Vector Of Excision Markers

Objective:Cre/loxP recombination system is the most wide application, and the specificity of the most studied restructuring system, To eliminate the influence of the non-purpose gene to genetically modified cells and animals, and have a better research to the single function of the inserted gene. using the Cre/loxP recombination system building a carrier that can erase marker gene, Building carrier based on the skeleton of commercial vector DsRed2-1which can produce specific business of red fluorescence, joined the LoxP sequence, and joined the MCS sequence which was advantageous to the purpose gene insertion,at the same time cloned the CMV promoter, synthesized by TK-IRES sequence, lay the foundations for the production of safer transgenic animals, for others to use these gene sequences to construct different uses of the carrier, laid a foundation for the needs of different experiments.Methods:(1)According to pIRES2-EGFP-CMV promoter cloned sequencing of CMV, the synthetic LoxP-MCS-LoxP sequences, successively connected to the Dsred2-1carrier to construct eukaryotic expression vector, named pCMV-Red-LoxP, synthetic Cre recombi-nase sequence, connected to a pTRE-tight carrier, and then connected tight-Cre fragments to pCMV-Red-LoxPcarrier, to build into a suspected pCMV-Tt-Cre-LoxP-Dsred carrier.(2) From plasmid pORF9-HSVltk cloned TK gene sequences, synthetic TK gene sequences, connect the TK gene sequences to pCMV-Red-LoxP carrier, named pCMV-Red-LoxP-TK, using well building vector transfect fine wool sheep fibroblasts, observed the results.(3) Using the restriction enzymes EcoRI and BamHI double digest pDsRed-CMV-LoxP-TK vector, connected the enzyme pieces to pEGFP-Cl carrier, named pEGFPCl-LoxP-CMV-TK, using the well built carrier and the carrier of enzyme fragment transfected respectively fine wool sheep fibroblasts.(4) Synthetized TK-IRES sequences, and a special synthetic sequence respectively connected to the built eukaryotic expression vector pCMV-Red-LoxP, named pCMV-LoxP-TK-IRES-Red2,using well built carrier transfected fine wool sheep fibroblasts with the electroporation method.(5) Connected the synthetic sequence of Cre recombinase to the pIRES2-EGFP carrier, named pIRES2EGFP-Cre, using well built vector transfect cells, extracted total RNA, RT-PCR method to detect, used the molecular level of Cre recombinase enzymes to cut the pCMV-LoxP-TK-IRES-Red2carrier,making restructuring reaction,using pCMV-LoxP-TK-IRES-Red2vector digested by the SmaI restriction enzyme,and then transfected into C2C12cells, G418screening positive clones, by pIRES2EGFP-Cre vector transfected into positive clone cells, observed statistics analysis, inducting positive clone cells with C oxygen guanosine (GCV), and observed statistical analysis.Results:(1) Successfully cloned the CMV promoter, constructed the suspected pCMV-Tt-Cre-LoxP-Dsred vector, identified two LoxP sequence between enzyme sites was disappeared, enzyme loci between two LoxP sequence was deleted.(2) Artificial TK gene sequence was synthesized successfully, a successful structuring pDsRed-CMV-LoxP-TK vector transfected into fine wool sheep fibroblasts, did not produce specific red fluorescence, but pDsRed-CMV-LoxP plasmid transfected into cells produced specific red fluorescence.(3) successfully constructed pEGFPCl-CMV-LoxP-TK, No enzyme and enzyme digestion of recombinant vector transfected into fibroblast cells at the same time, the not digested recombinant plasmid produced specific green fluorescence, but the digested did not produce specific green fluorescence.(4) Successfully built the pCMV-LoxP-TK-IRES-Red2vector, sent it to the biological company make sequencing, the results of sequence alignment showed the sequences of recombinant plasmid was correct, the recombinant vector by digested transfected into fiber cells using the electroporation method, produced the specificity of red fluorescence.(5) Successfully constructed pIRES2EGFP-Cre eukaryotic expression vector and it expressed the Cre recombinase, used the Cre recombinase The pCMV-LoxP-TK-IRES-Red2vector digested by enzyme has displayed recombination at the molecular level,the pCMV-LoxP-TK-IRES-Red2plastid transfected into C2C12cells, then obtained the red fluorescence positive cells screened by G418, the pIRES2-EGFP-Cre vector transfected into the positive clone cells, the ratio of the red fluorescent cells was reduced by24.3%, the GCV could effect to the positive clone cells and kill it, when the GCV could kill the positive clone cells but weak effected the natural cells, the critical concentration of GCV was between1μmol/L and10μmol/L.Conclusion:The expression vector built in this study can knockout marker genes. It has certain application value. The MCS could insert genes for different purposes, which laid a foundation for safer transgenic animal production. TK, IRES and LoxP gene could cut completely from pCMV-LoxP-TK-IRES-Red2,and laid a foundationfor other use.