STSM SCIENTIFIC REPORT
Carole LE POGAM CAGNOT
COST Action: BM0801
STSM title: Animal Model for MDS/AML transformation: application to the study of DNA vaccine.
Reference code: COST-STSM-ECOST--BM0801-120710-000000
Location:
School of Cancer & Imaging Sciences
The University of Manchester
Wolfson Molecular Imaging Centre
27 Palatine Road, Withington
Manchester
M20 3LJ
Host:
Pr Anthony Whetton
University of Manchester, School of Cancer and Imaging Center
[email protected]
Date of the mission:
July, 12th to 16th, 2010
Aim
Training on the CB-100 NanoPro (Cell Biosciences) and analysis of mouse samples to test the
hypothesis that DNA vaccine is able to influence the disease in our MDS model.
NanoPro principle
The CB-100 NanoPro is a nanofluidic proteomic assay allowing the separation of proteins from a very
small amount of cells. The proteins migrate in an electric field as long as the surrounding pH differs
from their pI. Once the pH = pI, the proteins are fixed in position (focused) regardless of applied
voltage. As post-translational modifications like phosphorylation can alter the actual pI, this assay
allows the study of the different phosphorylated forms of key signaling proteins.
Sample preparation: A minimum of 105 cells from freshly frozen spleen or peripheral blood are
thawed on ice, lysed in bicene chaps buffer and titrated (the final protein concentration for prepared
samples is somewhat assay-dependent, but should fall within the range of 0.025 - 0.10 mg/mL).
Protein extracts are mixed with the appropriate ampholyte premix and pI Standard Ladder (75%
Premix + pI ladder / 25% Lysate + Sample diluent buffer +inhibitor). Antibodies specific for the
studied proteins are diluted into an antibody diluents. Each preparation is then loaded into a specific
plate (example Fig. 1). The plate is then placed into the NanoPro.
Fig.1 : Example of a plate preparation
Run: The run consists of multiple sequential steps. These steps are controlled in Compass Software
through the use of several phases:
Load Phase: the head assembly moves to the resource tray, picks up twelve capillaries and
moves them to the Sample Tray. A slight vacuum is used to load samples from the sample plate
into the capillaries that are then transferred to the Separation Tray.
Separate Phase: The voltage is applied across the capillaries to drive the isoelectric focusing
separation of samples and pI standards. When the separation is complete, an image of the pI
standards is taken to determine the exact pI gradient profile, and a movie of the standards
separation is compiled. The capillaries are then exposed to UV light that activates a proprietary
linking chemistry on the inner wall of the capillaries, which links the separated proteins onto the
capillary walls. The capillaries then are rinsed with wash solution.
Primary Antibody Phase: the head assembly moves the capillaries to the sample plate, loads
primary antibody, and then transfers the capillaries to an Incubator Tray. When incubation is
complete, the capillaries are rinsed with wash solution.
Secondary Antibody Phase: the head assembly moves the capillaries back to the sample plate,
loads the secondary antibody, and then transfers the capillaries to an Incubator Tray. When
incubation is complete, the capillaries are rinsed with wash solution.
Detect Phase: the head assembly moves the capillaries back to the sample plate and loads the
luminol-hydrogen peroxide solution used for generation of chemiluminescence signal for the
sample proteins. The capillaries are then transferred to the Separation Tray where the emitted
chemiluminescent light is detected with a CCD camera. After capture of the chemiluminescent
image is complete, a final fluorescence image is recorded for precise registration of the sample
peak positions relative to the pI Standard Ladder positions.
Results Phase: indicates that results can be viewed in the Analysis screen.
Results:
• Nature of the cells for extraction
Comparing proteins from spleen or blood cells, we can observe that the major peaks are visible in
both spleen and blood extracts, but minor ones are only visible in spleen extracts. Optimization of
the protein extraction from blood is required (Fig. 2). Furthermore, it appeared that no consistent
peak was obtained from blood samples that have not been processed in a short time after the
bleeding (data not shown).
Spleen
Spleen
Spleen
Spleen
Fig. 2: Comparison of the signals obtained from spleen or white blood cells using an ERK 1/2 Ab.
• Comparison between placebo treated and DNA vaccinated MDS mice
Using the following antibodies, specific for different proteins of interest, spleen extracts from MDS
mice treated with either placebo (HBSS, buffer used as vehicle for the vaccine) or DNA vaccine have
been tested.
Antibodies tested:
- ERK 1/2
- ERK 1
- pERK 1/2
- Stat 5
- pStat 5
- CRKL
- panAKT
- pAKT(T)
- BCL-2
- pBCL-2
Despite migration problems, accurate analysis of the signals allowed us to observe some weak
differences between the placebo treated (HBSS) and DNA vaccinated (pVax14) mice. Using ERK 1
specific antibody, two small peaks, corresponding to ERK isoforms with pI of 5,3 and 7,5 are detected
in the extracts from DNA vaccinated mice (Fig. 3). These peaks are absent in the extracts from HBSS
treated mice, indicating that the vaccine has an effect on the RAS pathway. These peaks have to be
confirmed and identified in order to understand if the vaccine up- or downregulates this pathway.
Fig. 3: Peaks obtained
from spleen cells extracts
of MDS mice treated with
either placebo (HBSS) or
DNA vaccine (pVax14),
using an ERK 1 specific
antibody.
In the same way, traces obtained with a pBCL-2 specific antibody suggest differences in signals with
pI between 6,9 and 7,5. In this region, the peaks from the DNA vaccine samples are higher than the
ones from the HBSS treated mices (Fig. 4). Once again, these results have to be confirmed and
identified in order to interpret the effects of the vaccine on the BCL-2 pathway.
DNA Vaccine
HBSS
Fig. 4: Zoom on pBCL-2 peaks obtained with spleen cells extracts from MDS mice treated with either placebo (HBSS) or DNA
vaccine (pVax14).
Conclusions:
Further optimizations are needed for peak assessment, but these results gave clues on how the DNA
vaccine acts to maintain stable disease and increase survival of MDS mice. I obviously have to
increase the number of samples assayed. My mission in Manchester gave me the opportunity to be
trained on the CB1000 NanoPro machine and to acquire experience in designing assays and analyzing
results. I am now able to use the brand new NanoPro installed now in our Institute in order to
complete my studies using the samples I have frozen from my vaccination protocols during the last
year.
I thank the COST action BM0801 for having given me the opportunity to go to Manchester for this
training.