The Gateway

1
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.

2
MultiSite Gateway - Extending the applications
3
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

4
More att sequences needed
  CTGCTTTTTTGTACAAACTTG attB1   CAGCTTTCTTGTACA
AAGTTG attB2   CAACTTTATTATACAAAGTTG
attB3   CAACTTTTCTATACAAAGTTG
attB4   CAACTTTTGTATACAAAGTTG attB5
Standard Gateway
MultiSite Gateway
5
2-fragment MultiSite Gateway Pro
PCR Fragments
attB2
attB5
attB5r
attB1
X
X
X
X
pDONRs
attP2
attP5
attP5r
attP1
BP reactions
attL5
attL2
Entry Clones
X
attL1
attR5
X
Destination Vectors
attR2
attR1
LR reaction
Expression clones
attB2
attB5
attB1
6
3-fragment MultiSite Gateway Pro
PCR Fragments
attB4
attB1
attB3r
attB4r
attB2
attB3
X
X
X
X
X
X
pDONRs
attP4
attP1
attP3r
attP4r
attP2
attP3
BP reactions
attL4
attL1
attL2
attL3
Entry clones
X
X
attR3
attR4
Destination vector
attR2
attR1
CmR
ccdB
LR reaction
Expression clone
attB2
attB1
attB3
attB4
7
4-fragment MultiSite Gateway Pro
PCR Fragments
attB4
attB5
attB3r
attB4r
attB2
attB3
attB5r
attB1
X
X
X
X
X
X
X
X
pDONRs
attP4
attP5
attP3r
attP4r
attP2
attP3
attP5r
attP1
BP reactions
attL5
attL2
attL3
attL4
Entry Clones
X
X
X
attL1
attR4
attR3
attR5
X
Destination Vectors
attR2
attR1
LR reaction
Expression clones
attB2
attB4
attB3
attB5
attB1
8
MultiSite Gateway Three-Fragment Vector
Construction Kit
BP reactions
attR1
attL4
attL3
attR2
Entry clones
X
X
attL2
attL1
Destination vector
CmR
attR3
attR4
ccdB
LR reaction
9
Typical Results
Number of
Typical recombination
Expected colonies per
recombining
10 ?L reaction

ef
ficiency ()

fragments
1
10
-
10

90
-
100

3
6
3

10
-
10

70
-
90

3
4
4

10
-
10

30
-
80

2
3
10
In silico cloning using Vector NTI AdvanceTM 10.3
DNA of interest
Primers for PCR reaction
Cloning Strategy
11
Shortcomings when co-transfecting two plasmids
Plasmid 1
Plasmid 2
Courtesy of Dr. Imamoto, Osaka University, Japan

12
Example Expression of Multiple Genes in Human
Cells
CFP
YFP
A
B3
B2
CFP
pEF1
a
pCMV
B1
B4
YFP
a
B5
B3
B2
CFP
pEF1
B1
pCMV
B4
YFP
B
13
Rapid Testing of Expression Elements using
MultiSite Gateway
Kozak or
IRES
EGFP
Promoter
pABGH
HeLa
Kozak or
Gtx
Determination of expression level of EGFP
aurora A
2xGtx
cdc 2
5xGtx
cyclin B1
12xGtx
EMCV
cyclin E
IRES ( Internal Ribosome Entry Site )
mHCV2a
CMV
mHCV33
EF1-a
mHCV45
( CAG )
HCV2a
( SV40 )
HCV33
HCV45
Courtesy of Dr. Imamoto, Osaka University, Japan

14
Rapid Testing of Expression Elements using
MultiSite Gateway
Kozak or
Promoter
IRES
EGFP
pA
HeLa
Transcriptional signals with Kozak
Translational signals with CMV promoter
350
300
250
Relative activity
200
150
100
50
0
CMV
cdc 2
EF1-a
cyclin E
cyclin B1
aurora A
Courtesy of Dr. Imamoto, Osaka University, Japan

15
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