The Gateway Cloning System

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Invitrogen Proprietary & Confidential,*,The Gateway Cloning System,Cloning multiple fragments into a single vector,Contents,How to clone up to 4 DNA fragments simultaneously into one destination vector.,Examples of expression of multiple genes in HeLa cells.,Example of testing the effects of promoters and regulatory elements on protein expression.,MultiSite Gateway - Extending the applications,Your Application,Gene1,Gene2,Gene3,Gene4,Your Application,Gene,Protein,Localization,Gene,Gene,Protein,Purification,Gene,RNAi,Gene,Cell-Free,Gene,Protein,interaction,Gene,Gene,Entry Clone,PCR,Gene,synthesis,ORF,collection,Library,Your,Source,2,Invitrogen Proprietary & Confidential,Sample Applications,Optimized multigene delivery without co-transfection,Expression of enzymatic pathways,Expression of multi-subunit protein complexes,Gene knock-down and rescue (controllable RNAi and heterologous gene expression from the same construct),Variable gene expression levels using different expression elements,Combinatorial tagging,3,Invitrogen Proprietary & Confidential,CTGCTTT,TTTG,TACAAACTTG,att,B1,CAGCTTT,CTTG,TACAAAGTTG,att,B2,CAACTTT,ATTA,TACAAAGTTG,att,B3,CAACTTT,TCTA,TACAAAGTTG,att,B4,CAACTTT,TGTA,TACAAAGTTG,att,B5,Standard,Gateway,MultiSite,Gateway,More,att,sequences needed,4,Invitrogen Proprietary & Confidential,X,X,X,X,X,X,attB2,attB5,attP2,attP5,attR2,attR1,attB5r,attB1,attP5r,attP1,attL5,attL1,attB2,attB5,attB1,attL2,attR5,PCR Fragments,pDONRs,Entry Clones,Destination Vectors,Expression clones,2-fragment MultiSite Gateway Pro,BP reactions,LR reaction,5,Invitrogen Proprietary & Confidential,3-fragment MultiSite Gateway Pro,X,X,attB2,attB1,attB3,attB4,attL4,attL1,attL2,attL3,attR2,attR1,Entry clones,Destination vector,Expression clone,attR3,attR4,Cm,R,ccdB,attB4,attB1,attP4,attP1,X,X,attB3r,attB4r,attP3r,attP4r,X,X,attB2,attB3,attP2,attP3,X,X,PCR Fragments,pDONRs,BP reactions,LR reaction,6,Invitrogen Proprietary & Confidential,4-fragment MultiSite Gateway Pro,X,X,X,X,X,X,X,X,X,X,X,attB4,attB5,attP4,attP5,attB3r,attB4r,attP3r,attP4r,attB2,attB3,attP2,attP3,attR2,attR1,attB5r,attB1,attP5r,attP1,attL5,X,attL2,attL3,attL1,attB2,attB4,attB3,attB5,attB1,attL4,attR4,attR3,attR5,PCR Fragments,pDONRs,Entry Clones,Destination Vectors,Expression clones,BP reactions,LR reaction,7,Invitrogen Proprietary & Confidential,MultiSite Gateway Three-Fragment Vector Construction Kit,Cm,R,X,X,Entry clones,Destination vector,attB3,attB4,attB2,attB1,Expression clone,attR1,attL4,attL3,attR2,attR3,attR4,attL2,attL1,ccdB,X,X,X,X,attB1r,attB4,attP1r,attP4,attB2,attB1,attP2,attP1,attB3,attB2r,attP3,attP2r,X,X,PCR Fragments,pDONRs,BP reactions,LR reaction,8,Invitrogen Proprietary & Confidential,Typical recombination,Expected # colonies per,Typical Results,Number of,recombining,fragments,10 ,L reaction,ef,ficiency (%),2,10,3,-,10,5,80,-,100,3,10,3,-,10,4,70,-,90,4,10,2,-,10,3,30,-,80,1,10,3,-,10,6,90,-,100,9,Invitrogen Proprietary & Confidential,In silico,cloning using Vector NTI Advance,TM,10.3,Primers for PCR reaction,Cloning Strategy,DNA of interest,10,Invitrogen Proprietary & Confidential,Shortcomings when co-transfecting two plasmids,Plasmid 2,Plasmid 1,EGFP,P,CAG,mRFP,EGFP,mRFP,EGFP,mRFP,Courtesy of Dr. Imamoto,Osaka University, Japan,11,Invitrogen Proprietary & Confidential,Example: Expression of Multiple Genes in Human Cells,A,B,CFP,YFP,B1,B4,YFP,B3,B2,CFP,pEF1,a,pCMV,B5,B4,YFP,B3,B2,CFP,pEF1,a,B1,pCMV,12,Invitrogen Proprietary & Confidential,pA,BGH,EGFP,IRES,Promoter,Kozak or,HeLa,Determination of expression level of EGFP,IRES,(,Internal Ribosome Entry Site,),Kozak or,Gtx,2xGtx,5xGtx,12xGtx,EMCV,mHCV2a,mHCV33,mHCV45,aurora A,cdc 2,cyclin B1,cyclin E,CMV,EF1-a,(,CAG,),(,SV40,),HCV2a,HCV33,HCV45,Courtesy of Dr. Imamoto,Osaka University, Japan,Rapid Testing of Expression Elements using MultiSite Gateway,13,Invitrogen Proprietary & Confidential,pA,EGFP,IRES,Promoter,Kozak,or,HeLa,0,50,100,150,200,250,300,350,Relative activity,aurora A,cdc 2,cyclin B1,cyclin E,EF1-a,CMV,Transcriptional signals with Kozak,Translational signals with CMV promoter,Courtesy of Dr. Imamoto,Osaka University, Japan,7,13,1,1,29,13,9,4,7,0.0,5.0,10.0,15.0,20.0,25.0,30.0,35.0,40.0,None,Kozak,Gtx,2xGtx,5xGtx,12xGtx,EMCV,mHCV2a,mHCV33,mHCV45,Rapid Testing of Expression Elements using MultiSite Gateway,14,Invitrogen Proprietary & Confidential,Summary for MultiSite Gateway Technology,MultiSite Gateway Three-Fragment Vector Construction Kit,MultiSite Gateway Pro,Compatible with,Ultimate ORF clones,att,L1,-att,L2 entry clones,att,R4,-att,R3 DEST vectors,MultiSite Gateway Pro entry clones,att,R1,-att,R2 DEST vectors,Available for,Only 3-fragment cloning,2-, 3-, or 4-fragment cloning,Applications,Vector construction,Promoter analysis,Vector construction,Promoter analysis,Expression of multiple genes in one plasmid,Reporter analysis,and more,15,Invitrogen Proprietary & Confidential,