Microarray studies using mouse as model for the Human Disease
I. Overview
Clone sets
Experiment design
Gene expression profiles among selected mouse tissues
Mouse tissue, strain and sex variation
Rodent disease models
II. Preliminary data
Asbestos
TissueSexStrain: BrainSexStrain and LungSexStrain
Hypertension
LPS
SleepWake
Tissue-Tissue
Overview
In 2000, the National Heart , Lung and Blood Institute has launched a $37
million grant to establish 11 programs for Genomic Application aiming to
identify the human genes particularly relevant to heart, lung, blood, and
sleep functions. Mammals share many basic biological functions such as immune
response. Studies in species other than human may provide us with a
fundamental understanding of human diseases. Both the mouse and human
genomes contain about 3.1 billion base pairs. The gene sequence in human
and mouse encoding the functional proteins also share more than 85% identity.
Studies in rodent system could not only provide a powerful tool to interpret
the human genome, but also provide a valuable model system to study most of
the human diseases include the diseases related to the human heart, lung and
blood system.
Clone sets
The
main advantage of microarray technique is that it can assay differentially expressed
genes under parallel conditions. This
technique requires that the probes for the microarray should represent as
many (if not all) genes as possible for the specific tissue or species. In
2000, Ko et al developed the NIA
15k mouse clone set which represent 15,374 unique genes (78% novel and 22%
known at that time). We use this clone set in some of our microarray printing
for the study of gene expression profiles of strain and sex
variation in mouse brain cortex and brain stem as well as for LPS study (see below). In the later studies, we added another
Bmap
set (which has about additional 8k unique genes or EST) to the existing
NIA clone set to get NIA-Bmap clone set (which representing totally more than
23k unique genes or EST) for the microarray printing.
Experiment design
All
experiments are conducted using both loop and reference design. For the
reference design, we use the same pooled reference (pool all RNA samples from
the same experiment) for each specific experiment. Providing availability of RNA sample,
we use
reference experimental design, direct comparison, flip dye hybridization. Before the starting of this project we used
human cell lines and an array containing 9,600 human cDNA
clones spotted in duplicate to test the feasibility of conducting reference
loop designs (27 hybridizations)
Gene expression profiles among selected tissues
To find out tissue-tissue
differences in gene expression profile, the tissue surveys for mouse have been
conducting using the TIGR 27648-element mouse cDNA microarray based on the NIA and BMAP clone
set. Female mice of strain C57BL/6J
were sacrificed to collect total RNA from nine tissues (brain cortex(CO), brain
stem(BS), heart(HE), liver(LI), lung(LU), kidney(KI), intestine(INT), aorta(AO)
and reticulocytes(RE)). The "loop" and "reference"
experimental designs were employed to implement microarray hybridization by
following TIGR
protocol. phenotyping protocol Tissue-Tissue
Mouse tissue, strain and sex variation
We are generating an expression encyclopedia representing expressed sequences in
nine different tissues (Brain cortex, brain stem, lung, heart, kidney, aorta,
liver, intestine and reticulocytes). This study could provide us with overall information of gene
expression distribution in each tissue.
To increase our understanding of tissue and sexual differences in gene
expression in each tissue, Male and female mice of three different strains (
A/J, C57BL/6J
and DBA/2J)
were sacrificed to collect total RNA. We have
finished the gene expression profiling of brain cortex and brain stem,
and lung. Kidney will be our next target, with other tissues to be selected based on
available phenotypes. phenotyping protocol TissueSexStrain
Rodent disease models
- Asthma
Innate
Immunity can be assayed by studying immune response to pathogen challenge, or
modeled using response to stimulation by Lipo-polysaccharides (LPS). We are currently
collecting RNA samples from mouse mutants known to be either high or low
responders to LPS challenge, as well as the parental strains. phenotyping protocol Asthma-Innate_Immunity
- Asbestos disease model
Occupational
lung disease comprises a wide variety of disorders caused by the inhalation
or ingestion of dust particles or noxious chemical. These disorders include
pneumoconiosis, asbestos-related pleural and others. Although asbestos is
carcinogenic to humans, the mechanism by which it induces cancer is unknown.
The study from Brody AR
has shown that lung fibroblasts are activated to proliferate and produce
connective tissue during the development of lung fibrosis. The 129 mouse
strain does not develop asbestos-induced fibrogenesis, whereas several other
inbred strains rapidly respond to inhaled fibers. David Schwartz from Duke
University has bred C57B6 (high
responders) to 129 (low responder) and backcrossed the F1 mice to either C57B
or 129. RNA has been collected from lungs of both parental and progeny mice
and we are useing microarray to generate the gene express profile. The
purpose for this study is to find out the genes (either up-regulated or
down-regulated) involve in the inhalation of asbestos and provide a guideline
to the further study of human inhalation disease. phenotyping protocol Asbestosis
- Hypertension disease model
HypertensionTissueStrain
Hypertension may be induced in mice by introducing 1% salt into the drinking water of the mice.
The blood pressures of B6 and A/J mice drinking regular water are similar: however, those of B6
mice drinking 1% salt water increase by 15- 20 mm Hg, whereas those of A/J mice are unaffected
by 1% salt water. We will attempt to discover the genes responsible for this different blood pressure
response by comparing the gene expression patterns of both male and female B6 and A/J mice.
phenotyping protocol HypertensionTissueStrain
- Sleep and wakefulness
disease model
Control
of sleep and wakefulness is thought to be the result of two processes, the
circadian rhythm and sleep homeostasis. Even if there have been attempts to
study the changes in gene expression as a function of the cycle of sleep and
wakefulness, these efforts is far from comprehensive. Our collaborators in
the University of Pennsylvania
have conducted two complementary experiments. One is to determine the effect
of short-term (3 hours) of wakefulness, sleep deprivation (3 hours) and 3
hours of sleep. Another study will examine the effects of increasing
durations of sleep deprivation. RNA is collected from two brain regions:
cortex and hypothalamus. Our effort is using microarray technique to study
gene expression variation during different sleep and wakefulness stages so we
could have a best understanding of the functional roles of these genes in the
cycle of sleep and wakefulness or sleep deprivation. phenotyping protocol Prolonged Wakefullness
- The circadian rhythm
in gene expression in brain and peripheral organs
Many studies have demonstrate that there will be circadian variation in gene
expression in all peripheral tissues related to heart, lung and blood disease
as well as in cerebral cortex. Our collaborator in University
of Pennsylvania has proposed that
some of these circadian changes in gene expression will be similar in all
tissues, while other changes will be specific to different specific tissues
and reflect their more local metabolic activities. Our goal of this study is
to investigate this hypothesis. Allan I. Pack in UPenn will collect RNA from
mice during a full circadian cycle and we are ready to use them to assay
temporal patterns of gene expression.
- The effect of
intermittent hypoxia on gene expression in murine macrophages
Obstructive
sleep apnea (OSA) is condition in which people have repetitive airway
obstruction or hypopneas. Recent studies have demonstrated that OSA is an
important risk factor for cardiovascular disease including hypertension,
stroke and coronary artery disease. The elevations in blood pressure
associated with sleep apnea are independent of body mass, age and sex.
Additionally, even mild sleep apnea is associated with small increases in
blood pressure and severe sleep apnea is associated with more pronounced
hypertension. Researches in our group as well as in University
of Pennsylvania are using
microarray technique to evaluate the gene expression pattern using the RNA
collected from macrophages cultured under differently designed environmental
conditions: Intermittent hypoxia (mild, moderate, and severe); sustained
hypoxia; hypoxia/reoxygenation and normoxia. phenotyping protocol Intermittent Hypoxia
Rat Studies
