the gateway® cloning system
DESCRIPTION
Cloning multiple fragments into a single vector. The Gateway® Cloning System. Contents. How to clone up to 4 DNA fragments simultaneously into one destination vector. Examples of expression of multiple genes in HeLa cells. - PowerPoint PPT PresentationTRANSCRIPT
The Gateway® Cloning SystemCloning 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.
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MultiSite Gateway® - Extending the applications
Your Application
Gene1 Gene2 Gene3 Gene4
Your Application
GeneProtein
Localization
Gene
GeneProtein
Purification
Gene
RNAi
Gene
Cell-Free
GeneProtein
interaction
Gene
Gene
Entry Clone
PCR
Genesynthesis
ORFcollection
Library
YourSource
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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
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CTGCTTTTTTGTACAAACTTG attB1 CAGCTTTCTTGTACAAAGTTG attB2 CAACTTTATTATACAAAGTTG attB3 CAACTTTTCTATACAAAGTTG attB4 CAACTTTTGTATACAAAGTTG attB5
StandardGateway®
MultiSiteGateway®
More att sequences needed
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X XX X
X
X
attB2attB5
attP2attP5
attR2attR1
attB5rattB1
attP5rattP1
attL5
attL1
attB2attB5attB1
attL2
attR5
PCR Fragments
pDONRs
Entry Clones
Destination Vectors
Expression clones
2-fragment MultiSite Gateway® Pro
BP reactions
LR reaction
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3-fragment MultiSite Gateway® Pro
X X
attB2attB1 attB3attB4
attL4attL1 attL2attL3
attR2attR1
Entry clones
Destination vector
Expression clone
attR3attR4
CmR ccdB
attB4attB1
attP4attP1X X
attB3rattB4r
attP3rattP4rX X
attB2attB3
attP2attP3X X
PCR Fragments
pDONRs
BP reactions
LR reaction
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4-fragment MultiSite Gateway® Pro
X X X X X XX X
X X
X
attB4attB5
attP4attP5
attB3rattB4r
attP3rattP4r
attB2attB3
attP2attP3
attR2attR1
attB5rattB1
attP5rattP1
attL5
XattL2attL3
attL1
attB2attB4 attB3attB5attB1
attL4
attR4 attR3attR5
PCR Fragments
pDONRs
Entry Clones
Destination Vectors
Expression clones
BP reactions
LR reaction
Invitrogen Proprietary & Confidential8
MultiSite Gateway® Three-Fragment Vector Construction Kit
CmR
X XEntry clones
Destination vector
attB3attB4 attB2attB1Expression clone
attR1attL4 attL3attR2
attR3attR4
attL2attL1
ccdB
X X X XattB1rattB4
attP1rattP4
attB2attB1
attP2attP1
attB3attB2r
attP3attP2rX X
PCR Fragments
pDONRs
BP reactions
LR reaction
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Typical recombination Expected # colonies per
Typical Results
Number of recombining fragments
10 L reaction efficiency (%)
2 103-105 80-100
3 103-104 70-90
4 102-103 30-80
1 103-106 90-100
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In silico cloning using Vector NTI AdvanceTM 10.3
Primers for PCR reaction
Cloning Strategy
DNA of interest
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Shortcomings when co-transfecting two plasmids
Plasmid 2
Plasmid 1EGFPPCAG
mRFP
EGFP mRFP EGFP mRFP
Courtesy of Dr. Imamoto, Osaka University, Japan
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Example: Expression of Multiple Genes in Human Cells
A
B
CFP YFP
B1 B4YFP B3 B2CFPpEF1pCMV
B5 B4YFP B3 B2CFPpEF1B1 pCMV
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pABGHEGFPIRESPromoter
Kozak or
HeLa
Determination of expression level of EGFP
IRES ( Internal Ribosome Entry Site )
Kozak or Gtx
2xGtx 5xGtx 12xGtx EMCV mHCV2amHCV33 mHCV45
aurora A cdc 2
cyclin B1cyclin ECMVEF1-a( CAG )( SV40 )
HCV2aHCV33 HCV45
Courtesy of Dr. Imamoto, Osaka University, Japan
Rapid Testing of Expression Elements using MultiSite Gateway®
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pAEGFPIRESPromoterKozak or
HeLa
0
50
100
150
200
250
300
350
Rela
tive
act
ivit
y
auro
ra A
cdc
2
cycl
in B
1
cycl
in E
EF1-
a
CMV
Transcriptional signals with Kozak Translational signals with CMV promoter
Courtesy of Dr. Imamoto, Osaka University, Japan
713
11
29
139
4 7
0.05.0
10.015.020.025.030.035.040.0
Non
e
Koza
k
Gtx
2x
Gtx
5x
Gtx
12
xGtx
EM
CV
mH
CV2a
m
HCV
33
mH
CV45
Rapid Testing of Expression Elements using MultiSite Gateway®
Invitrogen Proprietary & Confidential15
Summary for MultiSite Gateway® Technology
MultiSite Gateway® Three-Fragment Vector Construction Kit
MultiSite Gateway® Pro
Compatible with… Ultimate™ ORF clones attL1-attL2 entry clones attR4-attR3 DEST vectors
MultiSite Gateway® Pro entry clones
attR1-attR2 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