The Gateway® Cloning System Cloning multiple fragments into a single vector Contents How to clone...
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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.
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Transcript of The Gateway® Cloning System Cloning multiple fragments into a single vector Contents How to clone...
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.
Invitrogen Proprietary & Confidential2
MultiSite Gateway® - Extending the applications
Your Application
Gene1 Gene2 Gene3 Gene4
Your Application
Gene
ProteinLocalization
Gene
Gene
ProteinPurification
Gene
RNAi
Gene
Cell-Free
Gene
Protein interaction
Gene
Gene
Entry Clone
PCR
Genesynthesis
ORFcollection
Library
YourSource
Invitrogen Proprietary & Confidential3
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
Invitrogen Proprietary & Confidential4
CTGCTTTTTTGTACAAACTTG attB1 CAGCTTTCTTGTACAAAGTTG attB2 CAACTTTATTATACAAAGTTG attB3 CAACTTTTCTATACAAAGTTG attB4 CAACTTTTGTATACAAAGTTG attB5
StandardGateway®
MultiSiteGateway®
More att sequences needed
Invitrogen Proprietary & Confidential5
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
Invitrogen Proprietary & Confidential6
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
Invitrogen Proprietary & Confidential7
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
X
attL2attL3
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
Invitrogen Proprietary & Confidential9
Typical recombination Expected # colonies per
Typical Results
Number of
recombining
fragments10 L reaction efficiency (%)
2 103-105 80-100
3 103-104 70-90
4 102-103 30-80
1 103-106 90-100
Invitrogen Proprietary & Confidential10
In silico cloning using Vector NTI AdvanceTM 10.3
Primers for PCR reaction
Cloning Strategy
DNA of interest
Invitrogen Proprietary & Confidential11
Shortcomings when co-transfecting two plasmids
Plasmid 2
Plasmid 1EGFPPCAG
mRFP
EGFP mRFP EGFP mRFP
Courtesy of Dr. Imamoto, Osaka University, Japan
Invitrogen Proprietary & Confidential12
Example: Expression of Multiple Genes in Human Cells
A
B
CFP YFP
B1 B4YFP B3 B2CFPpEF1pCMV
B5 B4YFP B3 B2CFPpEF1B1 pCMV
Invitrogen Proprietary & Confidential13
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®
Invitrogen Proprietary & Confidential14
pAEGFPIRESPromoterKozak or
HeLa
0
50
100
150
200
250
300
350
Rela
tive a
cti
vit
y
au
rora
A
cd
c 2
cyclin
B1
cyclin
E
EF1-a
CM
V
Transcriptional signals with Kozak Translational signals with CMV promoter
Courtesy of Dr. Imamoto, Osaka University, Japan
7
13
11
29
139
4 7
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
Non
e
Kozak
Gtx
2xG
tx
5xG
tx
12xG
tx
EM
CV
mH
CV
2a
mH
CV
33
mH
CV
45
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
