Cynthia B. Peterson, PI
Application/Protocol for Use of Live Vertebrates
A. Non-Technical Summary
Protocol for Breeding and Evaluation of Gene Expression in Wild-Type and
Knock-out Mice
Intensive efforts and abundant resources have been expended over the last few years to
determine the sequence of many genomes across the phylogeny, from microbes to mammals.
The database of information is rich, and these genomic resources now open the way for many
new approaches to biology in the “post-genomic” era. It is now important to annotate the
genome with information about the biological function of the individual gene products that are
encoded. For this reason, a variety of efforts across the scientific community are directed in this
new area of “functional genomics.” Experiments are aimed at understanding biology in terms
of networks of interacting genes and/or gene products. This proposal entails developing
methods to link genes to function using a “knockout” mouse as a model system. The long-term
goal is to develop methods that will allow for high-throughput screening using microarrays and
proteomics in conjunction with the mouse mutagenesis program at Oak Ridge National
Laboratory (ORNL). Microarrays, chips that contain thousands of genes (cDNAs) on a small
surface area, are state-of-the-art methodology for probing for expression of RNA corresponding
to these genes within various tissues. RNA is harvested form the tissue and labeled with
fluorescent probes that can be detected when bound to the cDNA targets on the chip.
Protoemics, on the other hand, is currently in development to test for the presence of the protein
products corresponding to these genes in a given tissue. For the proteomics approach, extracts
from a given tissue are separated by two-dimensional electrophoresis into thousands of “spots”
that correspond to the expressed proteins in the tissue. Mass spectrometry is then used to
identify which protein is found within a given “spot” on the two-dimensional gel according to
the unique mass identifier. We are currently working with researchers at ORNL to advance these
methods for screening and characterization of various mutant mice that are interesting models for
human disease.
1. Objectives:
a. develop a microarray chip that will be useful in evaluating expression of mRNAs in liver
and/or heart that correspond to circulating proteins in mouse plasma
b. To develop proteomics methods for analyzing levels of circulating proteins in mouse plasma
c. To use the microarray and proteomics methods to characterize differences at the genomic
level for a vitronectin -/- (knockout) mouse
d. To test whether changes to a high-fat diet affect the results using microarrays and proteomics
methods
2. Species and numbers of animals:
The vitronectin knockout mouse was generated at the University of Michigan by Dr. David
Ginsburg. Dr. Ginsburg has agreed to provide us with the knockout animals, and we will
work with Dr. O’Rourke and the animal facility staff in Walters Life Sciences to breed and
house the animals locally. The knockout mouse was generated on the C57/BL6 background,
so we plan to use the B6 strain as a normal control. The number of animals required was
derived by determining that 3-4 replicate experiments are required to establish
reproducibility of the data. We must compare differences between sexes. We also are
interested in correlating changes in gene and/or protein expression with aging, measuring
tissue-specific expression for newborns, weanlings, adult (~8 week) and old-age (~12-18
month) ages. We will breed cages with 4-5 animals to ensure that we have enough animals
for the different measurements.
Supplementary Information
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Cynthia B. Peterson, PI
Application/Protocol for Use of Live Vertebrates
3. Methods to reduce experimental stress or discomfort:
Animals will be anesthetized when whole blood samples are collected to provide material for
analysis of the protein expression (proteomics) from mouse plasma. They will also be
anesthetized prior to euthanasia at the time of tissue harvesting. Mice will be anesthetized by
inhalation of either isoflurane or metafane for the duration of the procedure. The primary
purpose of the anesthesia is to keep the animals still, and minimal stress and discomfort are
expected from these protocols.
4. Schedule of procedures:
a. Breeding: A breeding pair of animals has been obtained from the University of Michigan.
Mice are housed under standard conditions, according to AAALAC guidelines. Litters are
weaned at 3-4 weeks, depending on size. Complete records are kept on the mutant line. All
mice are inspected daily, and records are kept of birth and weaning dates.
b. Diet: A test group of animals will be fed a high-fat diet for 8 weeks and then the animals will
be harvested for testing.
c. Bleeding: For the proteome effort, circulating proteins in mouse plasma will be identified
and characterized using mass spectrometry measurements. Cutting-edge methods are being
developed that measure hundreds to thousands of different circulating proteins using a
combination of two-dimensional electrophoresis and mass spectrometry. The samples that
are needed to make these measurements are mouse plasma, which is isolated from whole
blood. This blood will be collected by orbital bleeds into capillary tubes that have been
treated with an anticoagulant. The blood is then centrifuged to remove the red blood cells,
and the remaining cell-free supernatant is the plasma. This procedure will only be conducted
twice per mouse, at 6 and 8 weeks of age. The amount of blood removed will be no more
than 200ul for a 20g mouse (within the 1% body weight guidelines established by OLAC).
d. Euthanasia and Surgery: Mice will be anesthetized with isoflurane as described above. The
animal is immediately sacrificed using cervical dislocation. Incisions are made in the lower
abdomen and chest cavity. The liver and heart are extracted and immediately frozen on dry
ice.
5. Benefits and expected outcome:
To date, the vitronectin knockout animal has no obvious “phenotype.” That is, the animals
develop and age normally, and they do not have spontaneous bleeding or clotting episodes.
When subject to certain stressors, however, the animals can exhibit altered coagulation behavior.
What effects does knocking out the gene for vitronectin have on the other genes that encode its
interacting partners in the circulation? Will other affected genes suggest phenotypic
abnormalities that have yet to be observed? What other interacting genes will be discovered
through a broad query? These interacting partners include not only the proteins mentioned
above, but also a potentially large set of unidentified gene products that would be revealed only
slowly by conventional biochemical methods. We are using state-of-the art technologies for
identifying all of the interacting genes and gene products from the set of thousands of genes that
are known from the mouse genome-sequencing project. We have set up arrays of 5000-10,000
genes that are tested for expression in the liver of the vitronectin knockout mouse. We now
intend to compare animals on normal diets to those on a high-fat diet because of the compelling
evidence that links vitronectin to cardiovascular disease. This is known to be correlated with a
high-fat diet in humans. Also, our collaborators have recently detected vitronectin in the adipose
tissue of obese humans. This gene expression data with the under normal and high-fat diets will
be complemented with protein expression (proteomics) data that uses two-dimensional
Supplementary Information
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Cynthia B. Peterson, PI
Application/Protocol for Use of Live Vertebrates
electrophoresis and mass spectrometry to identify proteins that are expressed at abnormal levels
in the circulation of the knockout mouse.
Increasing the scope of our query from the known set of proteins that have been shown to
interact with vitronectin to the genome-wide scale is important in this new age of functional
genomics. Indeed, the National Heart, Lung and Blood Institute (NHLBI) recently identified the
need for the research community to develop genomic resources for scientists and clinicians
interested in function and dysfunction in hematology, pulmonary and cardiovascular function. A
formal call for proposals for centers interested in providing these types of resources was
announced by NHLBI, and our laboratory, as a part of the Tennessee Mouse Genome
Consortium (TMGC), responded to the call. The TMGC proposal was focused on the
application/development of animal models for human disease. The TMGC is an affiliation
between Oak Ridge National Laboratory, the University of Tennessee, Knoxville, Vanderbilt
University, MeHarry School of Medicine, the University of Memphis, the University of
Tennessee Medical Center at Memphis, and St. Jude’s Research Institute. One of the original
emphases for the consortium at its conception was the development of high-throughput screens
for a wide variety of altered phenotypes in mice produced from the ORNL mutagenesis program.
Accordingly, the TMGC proposed to screen the test class animals for alterations in hematologic
and cardiovascular function. Although our proposal was viewed favorably upon peer review, it
was not one of the few proposals (10) funded across the country. Now we are in the process of
revising the proposal so that it is focused on particular problems of interest to the researchers in
the TMGC. This pilot project will provide us with the opportunity to gather some data on a
model system that will serve as a “proof of principle” for our plans to implement the microarray
and proteomics screens in a high-throughput fashion to characterize hundreds of mutant mice
generated in the mammalian genetics program at ORNL.
Supplementary Information
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