TVDC Progress Report
for ASU
8/28/07
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25 and 32
Active Milestones: 26, 28, 33, 34, 35
Currently Inactive Milestones: 30, 36-38
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Slide 1
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 33
Printing and testing
GDP confirmed
Printing arrays
Gray: (sub )milestone title
Red: completed
Green: in progress
GDP Confirmation
Comparisons of substrate
Poly-L Lysine vs Corning Ultragaps
Compare TIGR PFGR Arrays to in house arrays
Testing of linear amplification of procaryotic
Transcripts (LAPT) process and dilution testing of
Schu S4 RNA with and without mouse lung RNA
RNA shipped 1/29/2007
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Slide 2
ASU-TVDC
INNOVATIONS IN MEDICINE
Re-design of Amplification Process
• Clontech cDNA synthesis kit reformulated without RT enzyme
• Need a replacement enzyme with Terminal Transferase activity
without RNAse H activity
• InVitrogen SuperScript II (not SuperScript III)
Amplification Yield after LAPT (total micrograms)
g SCHU S4 amplified
1.0
0.1
0.0
SSII-SSII Buffer
4
9.3
13.8
SSIII-SSIII Buffer
6.2
6.3
Not done
SSII-Clontech Buffer
24.2
31.3
15.7
• Clonetech buffer had final 6 mM MgCl2 concentration where as
InVitrogen’s buffer had 3 mM
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Slide 3
ASU-TVDC
INNOVATIONS IN MEDICINE
Modification of Buffer Systems
•
•
Additional potential modifications included an addition of 2 mM MnCl2 for
the last 20 minutes of the cDNA synthesis
Set up a comparison of InVitrogen buffer with supplemented MgCL2 with
and without MnCl2 to amplify 0.1 g SCHU S4 in 1 g normal mouse lung
RNA
Amplification Yield after LAPT (total micrograms)
mM MgCl2
3
6
9
No MnCl2
7
40
65
With MnCl2 (2mM)
29
54
67
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Slide 4
ASU-TVDC
INNOVATIONS IN MEDICINE
Top 300 Genes
Amplified 0.1 g
6mM MgCl2
Average of 3 Previous
0.1 g LAPT Samples
Group
131
36
44
106
28
113
53
1299
Pearson
Coefficient
0.1 Average
v 6 mM MgCl2
.455
0.1 Average
v 9 mM MgCl2
.437
6 mM MgCl2
v 9 mM
.872
Amplified 0.1 g
9mM MgCl2
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Slide 5
ASU-TVDC
INNOVATIONS IN MEDICINE
Milestone 33 Summary
• Amplification system working
• Need to determine if MnCl2 addition is beneficial
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Slide 6
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 34
Pilot studies of optimization of RNA
isolation and hybridization conditions
Gray: (sub )milestone title
Red: completed
Green: in progress
RNA Isolation (UNM
Hybridization Conditions
Initial testing of heavily infected lungs
Perform CFU analyses and compare with purified RNA
Testing Maui Hybridization chamber
Amplification testing of Schu S4 RNA
with and without mouse lung RNA
RNA isolated from infected lungs received from UNM
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Slide 7
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Gray: (sub )milestone title
Red: completed
Green: in progress
Virulent Schu 4 Samples
RT-PCR Confirmations
Initial samples
Dose-Response of Infection
To Be Determined
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Slide 8
ASU-TVDC
INNOVATIONS IN MEDICINE
Milestone 35 Summary
• Received dose response samples from
UNM
• Performed RNAeasy purification
– Purified individual samples
– 3 mice per group
– 103, 104, 105, 106, 107 FTU/Mouse
• Made Pool of each dose
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Slide 9
ASU-TVDC
INNOVATIONS IN MEDICINE
Amplification Profile of Dose Response Challenge
Infection dose
g after LAPT
# Genes >2 fold to 0
0
18
-
103
5
192
104
42
260
105
51
197
106
30
190
107
42
538
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Slide 10
ASU-TVDC
INNOVATIONS IN MEDICINE
Overlap of Genes Identified After Amplification
103
104
67
30
9
86
61
105
104
72
96
41
43
46
81
172
47
23
159
43
105
106
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Slide 11
ASU-TVDC
INNOVATIONS IN MEDICINE
Upcoming Monthly Transcriptome Goals
• Repeat previous mouse samples with new amplification
process
• Dose response curve with new amplification process
• Test individual samples of animals challenged with varying
doses of SCHU S4 9
– Repeat dose response 107 – 10 (include mock challenge = 0)
• Amplification samples to be retested on TIGR and ASU
arrays
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Slide 12
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 26
Prepare a highthroughput protein
production system
Gray: (sub )milestone title
Red: completed or inactive
Green: in progress
Test ORF synthesis
and select expression
constructs
Select and test
IVT Protocols
Select and test protein
purification protocols
Expression templates
further optimized as needed
for purification
in vitro protein yields
are optimized
Purified vs. unpurified samples
are ready for being at NM
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Slide 13
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1
Test ORF synthesis and select expression
constructs
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Slide 14
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Previous Status
• A modular system allowing quick modification of the existing
IVT cassette has been developed.
• Use of the raw gene assembling reaction as an IVT template
yielded same quality and quantity of the IVT products as a
perfectly assembled clone equivalent.
• LEE production will start upon completion of the above
evaluation and the ongoing T-cell stimulation experiments.
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Slide 15
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Current Status
728b Pool
1434a Pool
728a Pool
1434a Clone
728b Clone
728a Clone
728a Clone
728a Pool
728b Clone
728b Pool
1434a Clone
The earlier reported IVT yield differences are due to the
variable quality of the used PCR template.
1434a Pool
•
30 26 29 26 7 21 ug
DNA agarose gel
Protein SDS-PAGE
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Slide 16
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Current Status
•
Optimization of the LEE assembling conditions
– Effect of His tag presence and location
Dbl His
By product
N His
C His
No His
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Slide 17
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Current Status
•
Optimization of the LEE assembling conditions
– Effect of template concentration and polymerase (Taq, iProof)
Blue – Taq polymerase
Red – iProof polymerase
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Slide 18
ASU-TVDC
INNOVATIONS IN MEDICINE
Conclusions
• Simple PCR purification and quantification of PCR product is
not sufficient for template evaluation. At least e-gel evaluation
should be implemented for QC of IVT templates.
• Conditions have been worked out for tag incorporation into IVT
products.
• Optimal number of cycles has been determine to accommodate
best possible quality of template.
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Slide 19
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 2
Select and test IVT Protocols
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Slide 20
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 2. Previous Status
• Applied in HTP format, the developed protocol reliably
generates > 20ug of protein per reaction.
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Slide 21
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 2. Current status
Test impact of shaking on IVT product yields
CPM Total
CPM #met
ug prot
1
FTU 1419 300 rpm
561,200
140,300
15.55
2
FTU 1419 300 rpm
547,520
136,880
15.17
3
FTU 1419 400 rpm
592,200
148,050
16.41
4
FTU 1419 400 rpm
606,960
151,740
16.82
5
FTU 1419 800 rpm
1,133,760
283,440
31.41
6
FTU 1419 800 rpm
952,280
238,070
26.39
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Slide 22
ASU-TVDC
INNOVATIONS IN MEDICINE
Conclusions
• Shaking IVT reaction less than 600rpm reduces IVT protein
yields.
• Regular tissue culture shaker is not suitable for optimal IVT
reaction conditions. The existing ProteoMaster machine
(Roche) handles only one 96-well plate at the time.
• Several high speed shakers will need be purchased to
accommodate throughput.
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Slide 23
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 3
Select and test protein purification protocols
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Slide 24
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim. 3 Previous Status
• The current protocol is associated with significant loses
during acetone precipitation and re-solubilization of the IVT
reaction products.
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Slide 25
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim. 3 Current status
• Ammonium sulfate fractionation as an alternative to acetone
precipitation
0%
5 10 20
25% 50% 75% 90%
S
P S P S P S P
0%
5 10 20
25%
S
P
• Novel IVT proteins are mostly precipitated by 25% ammonium sulfate
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Slide 26
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim. 3 Current status
•
Effect of detergents on protein solubility
dH2O
20% DMF
3% CHAPS
1%Deoxychola
te
FTU 1434a
8294
NA
396
15466
FTU 728a
4620
352
5874
NA
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Slide 27
ASU-TVDC
INNOVATIONS IN MEDICINE
Conclusions
•
•
•
Ammonium sulfate fractionation cannot be used as an
alternative to acetone precipitation. There is no discriminating
range for selective precipitation of the novel or bacterial
proteins.
Use of nonionic detergents can help solubilization of novel IVT
proteins, but none of those tested to date works universally.
Further studies are needed to address this issue.
We found inconsistency in the quality and performance of the
Ni-beads. His purification experiments will be repeated to
confirm earlier conclusions.
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Slide 28
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 28
Build SCHU S4
proteome
Gray: (sub)milestone title
Red: inactive
Green: in progress
Build ORF expression
library corresponding
to proteome
Generate complete
protein-fragment library
Array protein-fragments
into measurable pools
For T cell stimulation
W.T. ORFs
will be finished
In ~1 week
Inactive
Inactive
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Slide 29
ASU-TVDC
INNOVATIONS IN MEDICINE
Build ORF expression library
corresponding to proteome
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Slide 30
ASU-TVDC
INNOVATIONS IN MEDICINE
Previous Status
•ORF amplification protocols are in place.
•Templates and primers for promoter and terminator
amplification have been tested and readily available.
•Database management and tracking system is in place.
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Slide 31
ASU-TVDC
INNOVATIONS IN MEDICINE
Current Status
• Agilent chips have been received.
• Oligomix modifications are being done (phosphates)
• Gene building protocol optimizations have begun
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Slide 32
ASU-TVDC
INNOVATIONS IN MEDICINE