Transcript Cloning multiple fragments into a single destination vector

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
PCR
Your
Source
Library
ORF
collection
Gene
synthesis
Gene
Gene
Gene
Protein
Localization
Your Application
Gene
Entry Clone
Gene
Gene
Protein
Purification
RNAi
Gene
Gene
Cell-Free
Protein
interaction
Gene1
Gene2
Gene3
Gene4
Your Application
2
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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

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Combinatorial tagging
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More att sequences needed
Standard
Gateway®
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CTGCTTTTTTGTACAAACTTG
attB1
CAGCTTTCTTGTACAAAGTTG
attB2
CAACTTTATTATACAAAGTTG
attB3
CAACTTTTCTATACAAAGTTG
attB4
CAACTTTTGTATACAAAGTTG
attB5
MultiSite
Gateway®
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2-fragment MultiSite Gateway® Pro
PCR Fragments
pDONRs
attB1
attB5r
attB5
attB2
X
X
X
X
attP1
attP5r
attP5
attP2
BP reactions
attL5
Entry Clones
attL2
X
attL1
attR5
X
Destination Vectors
attR1
attR2
LR reaction
Expression clones
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attB1
attB5
attB2
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3-fragment MultiSite Gateway® Pro
PCR Fragments
pDONRs
attB1
attB4
attB4r
attB3r
attB3
attB2
X
X
X
X
X
X
attP1
attP4
attP4r
attP3r
attP3
attP2
BP reactions
Entry clones
Destination vector
attL1
attR1
attL4
attL3
X
X
attR4
attR3
CmR
ccdB
attL2
attR2
LR reaction
Expression clone
6
attB1
attB4
attB3
attB2
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4-fragment MultiSite Gateway® Pro
PCR Fragments
pDONRs
attB1
X
attP1
attB5r
attB5
X
attP5r
attB4
attB4r
attB3r
X
X
X
X
attP5
attP4
attP4r
attP3r
attB3
attB2
X
X
attP3
attP2
BP reactions
attL5
Entry Clones
attL1
attL4
attL3
X
X
X
attR5
attR4
attR3
attL2
X
Destination Vectors
attR1
attR2
LR reaction
Expression clones
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attB1
attB5
attB4
attB3
attB2
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MultiSite Gateway® Three-Fragment Vector Construction Kit
PCR Fragments
pDONRs
attB4
attB1r
attB1
attB2
attB2r
attB3
X
X
X
X
X
X
attP4
attP1r
attP1
attP2
attP2r
attP3
BP reactions
Entry clones
Destination vector
attL4
attR4
attR1
attR2
X
X
attL1
attL2
CmR
ccdB
attL3
attR3
LR reaction
Expression clone
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attB4
attB1
attB2
attB3
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Typical Results
Number of
recombining
fragments
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Expected # colonies per
10 L reaction
Typical recombination
efficiency (%)
1
103-106
90-100
2
103-105
80-100
3
103-104
70-90
4
102-103
30-80
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In silico cloning using Vector NTI AdvanceTM 10.3
DNA of
interest
Primers
for PCR
reaction
Cloning
Strategy
10
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Shortcomings when co-transfecting two plasmids
EGFP
mRFP
PCAG
EGFP mRFP
EGFP
Plasmid 1
mRFP
Plasmid 2
Courtesy of Dr. Imamoto, Osaka University, Japan
11
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Example: Expression of Multiple Genes in Human Cells
CFP
A
B
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pCMV
B1
B1 pCMV B5
B4 pEF1 a B3
YFP
YFP
B4 pEF1 a B3
CFP
CFP
YFP
B2
B2
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Rapid Testing of Expression Elements using MultiSite Gateway®
Kozak or
IRES
Promoter
EGFP
pABGH
HeLa
aurora A
cdc 2
cyclin B1
cyclin E
CMV
EF1-a
( CAG )
( SV40 )
Kozak or
Gtx
2xGtx
5xGtx
12xGtx
EMCV
mHCV2a
mHCV33
mHCV45
HCV2a
HCV33
HCV45
Determination of expression level of EGFP
IRES ( Internal Ribosome Entry Site )
Courtesy of Dr. Imamoto, Osaka University, Japan
13
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Rapid Testing of Expression Elements using MultiSite Gateway®
Kozak or
Promoter IRES
EGFP
pA
HeLa
Transcriptional signals with Kozak
40.0
350
35.0
300
29
30.0
250
10.0
50
5.0
1
1
5xGtx
2xGtx
Gtx
Kozak
None
EF1-a
CMV
cdc 2
aurora A
cyclin E
0.0
0
9
4
7
7
mHCV45
100
13 13
EMCV
15.0
12xGtx
150
mHCV2a
20.0
mHCV33
25.0
200
cyclin B1
Relative activity
Translational signals with CMV promoter
Courtesy of Dr. Imamoto, Osaka University, Japan
14
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Summary for MultiSite Gateway® Technology
MultiSite Gateway® ThreeFragment Vector Construction
Kit
Compatible with…
 Ultimate™ ORF clones
 attL1-attL2 entry clones
MultiSite Gateway® Pro
 MultiSite Gateway® Pro entry
clones
 attR4-attR3 DEST vectors
 attR1-attR2 DEST vectors
Available for…
 Only 3-fragment cloning
 2-, 3-, or 4-fragment cloning
Applications
 Vector construction
 Vector construction
 Promoter analysis
 Promoter analysis
 Expression of multiple genes
in one plasmid
 Reporter analysis
…and more
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