Transcript P G A ROGRAMS

PROGRAMS FOR
GENOMIC
APPLICATIONS
National Heart, Lung, and Blood Institutes
National Institutes of Health
Mission Statement
To develop new resources, reagents,
and education programs for
investigators engaged in NHLBIrelated research.
PGA Mission
• Provide new resources and reagents to link
genes to biological function and make these
readily available to the NHLBI community.
• Facilitate workshops, courses, and visiting
scientist programs to train investigators in the
technologies being applied in the PGAs.
• Rapidly disseminate data through the world
wide web and public databases.
Organizational Structure
Coordinating Committee
Bioinformatics
Subcommittee
Phenotype
Subcommittee
Proteomics
Subcommittee
Microarray
Subcommittee
Data Sharing
Subcommittee
Integration
Subcommittee
Education
Subcommittee
PGA Programs
Applied Genomics in CardioPulmonary Disease
Johns Hopkins University School of Medicine
Genomics of Cardiovascular Development,
Adaptation, & Remodeling
Harvard Medical School
Physiogenomics of Stressors in Derived
Consomic Rats
Medical College of Wisconsin
Mouse Models of Heart, Lung, and Blood
Diseases
The Jackson Laboratory
Expression Profiling of Rodent Models of
Human Disease
The Institute for Genomics Research
Comparative Genomic Analysis of
Cardiovascular Genes
Genomics of Proteomics of Cell Injury and
Inflammation
Lawrence Berkeley National Laboratory
University of Texas S.W. Medical Center
Harvard Medical School
Innate Immunity in Heart, Lung, and Blood
Diseases
NHLBI Bay Area Functional Genomic
Consortium
The University of Arizona
The David J. Gladstone Institute
UW-FHCRC Variation Discovery Resource
University of Washington
Genomic Analysis of Stress and Inflammation
Bioinformatics
• Carol Bult, Ph.D., The Jackson Laboratory
Data Sharing
• Isaac Kohane, M.D., Ph.D., Harvard Medical School
Subcommittee
Chairs
Education
• Scott Weiss, M.D., M.S., Harvard Medical School
Genomic Inventory/Integration
• Edward Rubin, M.D., Ph.D., The Lawrence Berkeley
National Laboratory
Microarray
• John Quackenbush, Ph.D., The Institute for Genomics
Research
Phenotype
• Andrew Greene, Ph.D., Medical College of Wisconsin
Proteomics
• Thomas Kodadek, Ph.D., Univ. Texas S.W. Medical Center
Anticipated PGA Resources/Tools
•
Mouse models of HLBS disorders
•
Rat models of HLBS disorders
•
Microarrays
•
DNA Variations (SNPs - locations, allele
frequencies, genotypes and haplotypes)
•
Reagents (clones, antibodies, mice, and rats)
•
Protocols
•
Bioinformatic Resources (software tools and
databases)
BayGenomics
http://baygenomics.ucsf.edu
Focus: Cardiopulmonary Development and Disease
•
Apply custom gene-trap vectors to inactivate genes in
ES cells and to evaluate the functional importance of
these in cardiopulmonary development and disease
using computational approaches, expression profiling,
in situ hybridization studies, and in select cases in
animals.
PI: Dr. Stephen G. Young
CardioGenomics
http://www.cardiogenomics.org
Focus: Cardiovascular Development, Adaptation, and
Remodeling
•
To link genes to function, dysfunction, and structural
abnormalities of the cardiovascular system caused
by clinically relevant genetic and environmental
stimuli.
PI: Dr. Seigo Izumo
HopGenes
http://www.hopkins-genomics.org
Focus: Tissue Remodeling in Cardiopulmonary Disease
•
To identify the genes involved in tissue remodeling using
expression profiling to explore the pathology of asthma,
chronic obstructive pulmonary disease, cystic fibrosis, lung
transplantation, acute lung injury, scleroderma, sarcoidosis,
pulmonary hypertension, ischemic cardiomyopathy, and
cardiac transplantation.
PI: Dr. Joe G.N. Garcia
Innate Immunity
http://innateimmunity.net
Focus: Genetics of HLB Disorders
•
Explore genetic susceptibility in asthma, chronic
obstructive pulmonary disease, myocardial infarction
and deep venous thrombosis by evaluating
polymorphisms in genes involved in innate immune
responses.
PI: Dr. Fernando D. Martinez
JAX PGA
http://pga.jax.org
Focus: Mouse Models of HLBS Disorders
•
Apply a phenotype-driven approach to identify
the genetic mechanisms underlying the
physiology and pathophysiology of
atherosclerosis, hypertension, lung function,
blood formation, thrombosis, obesity,
inflammation, and sleep function.
PI: Dr. Luanne L. Peters
PARABIOSYS
http://genetics.mgh.harvard.edu/Parabiosys/
Focus: Genetics of Inflammation and Stress
•
To identify and characterize the gene networks
activated by pro-inflammatory, metabolic, and
pathogenic stresses affecting cardiovascular
and pulmonary systems.
PI: Dr. Brian Seed
Berkeley PGA
http://pga.lbl.gov
Focus: Cardiovascular Gene Expression
•
Apply comparative genomics to identify and
understand the role of cis-acting regulatory
elements that affect the expression of
cardiovascular genes.
PI: Dr. Edward M. Rubin
PhysGen
http://pga.mcw.edu
Focus: Rat Models of HLBS Disorders
•
Dissect multigenic common HLBS
diseases through the development of
panels of chromosomal substitution strains
of rats (consomic rat panels).
PI: Dr. Howard J. Jacobs
Seattle SNPs
http://pga.mbt.washington.edu
Focus: Inflammation and Genetic Variability
•
To identify variable sites in human genes to expand
the resources available to explore the role of interindividual variation and its relationship to disease
risk, outcome and treatments for common human
disorders.
PI: Dr. Deborah A. Nickerson
Southwestern
http://pga.swmed.edu
Focus: Cell Injury and Inflammation
•
Elucidate the basic mechanisms underlying
cell injury and inflammation through a
combination of genomic and proteomic
approaches.
PI: Dr. Stephen A. Johnston
TREX
http://pga.tigr.org
Focus: Gene Expression in HLBS Disorders
•
Explore gene-environment interactions using rodent
models of human disease and cDNA microarray
assays to elucidate patterns of gene expression in
heart, lung, blood, and sleep disorders.
PI: Dr. John Quackenbush
NHLBI PGA Research Network
• SeattleSNPs - Seattle
.
• PhysGen - Milwaukee
.
• BayGenomics
..
• Berkeley PGA
- San Francisco
- Berkeley
.
.
• InnateImmunity
- Tucson
• Southwestern - Dallas
. • JAX PGA . •• CardioGenomics
ParaBioSys
Bar Harbor
Boston
- Boston
..
• HopGenes - Baltimore
• TREX - Rockville
NHLBI PGA Web Sites
PROGRAMS FOR GENOMIC APPLICATIONS
http://www.nhlbi.nih.gov/resources/pga/index.htm
PGA Web Sites (cont.)
BayGenomics - http://baygenomics.ucsf.edu
CardioGenomics - http://www.cardiogenomics.org
HopGenes - http://www.hopkins-genomics.org
InnateImmunity - http://innateimmunity.net
JAX PGA - http://pga.jax.org
ParaBioSys - http://genetics.mgh.harvard.edu/Parabiosys/
PGA Web Sites (cont.)
Berkeley PGA - http://pga.lbl.gov
PhysGen - http://pga.mcw.edu
SeattleSNPs - http://pga.mbt.washington.edu
Southwestern - http://pga.swmed.edu
TREX - http://pga.tigr.org
RAT GENOME
SEQUENCING PROJECT
Baylor College of Medicine
NHGRI/NHLBI
Howard Jacob
MCW
Marco Marra
UBC
Shaying Zhao
TIGR
Peter Tonellato
Rat GDB
CHO
Pieter de Jong
Univ Utah
Robert Weiss
Goals
Produce a `draft sequence` of the genome,
Assembly and first-pass annotation,
Cross-species comparison,
A public assembler,
Foundation for a finishing effort,
Interface with community,
Fold-Coverage Timelines (Year One)
(Cumulative)
Time
Q1
Q2
Q3
Q4
BCM
Celera
0.25/0.13 0.28/0
0.59
0.56
0.98
0.84
1.36
1.12
GTC
Total
0.07/0.21
0.53
0.14
1.15
0.21
1.82
0.28
2.48
All in MOU (with data release policy)
Cumulative Sequencing Schedule
3.00
2.50
BCM
BCM projected
Coverage on 3 Gbp
2.00
GTC
GTC projected
Celera projected
RAT target
1.50
1.00
0.50
0.00
31-Oct-00
30-Jan-01
1-May-01
31-Jul-01
31-Oct-01
30-Jan-02
Strategy: Strain Selection:
Howard Jacob’s BN/SsNHsd
This Brown Norway strain has only
seven heterozygous markers in the
more than 4,000 tested i.e. it is very
`isogenic` as a representative strain.
New BAC Library
Made by Pieter de Jong from BN/SsNHsd female,
One enzyme so far
~200 kb inserts
10 X coverage
Second enzyme to come?
Doug Smith pursuing chr Y library,
Fingerprints:
UBC, ~40,000 for `de-randomization`
~200,000 for f`print assembly,
BES:
TIGR, ~200,000 paired ends
Strategy: BAC Skims
BAC skims to cover 90% of genome
Originally 20,000 150 kb `de-randomized`
BACs @ 1.0 x coverage,
(384 reads per BAC) = 1 x clone coverage
= 67% - 80% coverage
~ 5,000 gaps for walks,
Now 15,000 200 kb BACs @768 reads
per BAC and 1.7 X coverage
Strategy: BAC Skims (cont`d)
Reads in BACs:
2kb paired end (dye term) +++
10kb paired end (dye term) -M13 (with primer chemistry) ?
Strategy: WGS Reads
2kb paired end (dye term) +++
10kb paired end (dye term) +++
5kb and 50kb discussed,
M13 (with primer chemistry) ?
Challenges:
- Usual
- Assembly
- Data `purity`
Sequencing Reads
- Whole Genome Shotgun
- Small insert (1-3 kb)
- Large insert (10 kb)
- BAC skims
- BAC ends
Atlas:
Assemble
Within Bins
Phrap
Sequence Comparison
Sequence Comparison
- Sampling
- Suffix Tree
A whole genome
assembler
Initial Bin 1
- Consensus Sequence
- Unused reads
Find Overlapping
Sequences
Find Overlapping
Sequences
- Breadth first binner
- Breadth first binner
Initial Bin 2
Initial Bin 3
Join Bins
Join Bins
Containing
Unique BAC
Skim Reads
High
Confidence
Bin 1
High
Confidence
Bin 2
High
High
Confidence
Confidence
Composite Bins
Composite Bins
Add Available
Paired Ends to
Bins
- Sequence Overlap
- Sufficient End Pairs
High
Confidence
Bin 3
Iterate While
Significant New
Data is Added
To Finishing
Group
Genomics Component
Genotyping & Animal Production
(Transponders and transfer)
2,640
15 strains/yr
Phenotyping Component
Conditioning Protocol
Respiratory
Studies
high salt & salt depletion
Systemic Vascular
& Renal Studies
hypoxia
normoxia
Lung Studies
hypoxia
normoxia
Cardiac Studies
low salt
Isolated Vessel
Blood Studies
Genomics
Component
Research Services Component
Bioinformatics
Consomic Rat
Strains
Rat
distribution
Functional
Mapping
Component
Baseline & Stressed
Physiological Data
high salt
Baseline & Stressed
Expression Data
Expression
Profiling
TIGR Consomic
Expression Data
DNA & Genotyping
Data
Phenotyping Team
Mary Pat Kunert, RN, Ph.D., Project Supervisor
Mindy Dwinell, Ph.D. Project Supervisor
Meredith Skelton, M.S. , Sr. Research Associate
Julie Messinger, M.S., Program Coordinator
Cardiac Studies:
Jessica Laessig and William Hutchins
Renal Studies:
Larry Clowry and Mike Bregantini
Biochemistry:
Candace Jones
Lung Studies:
Jess Powlas and Bernadette Cabigas
Isolated Vessel Studies:
Janelle Yarina
Respiratory Studies:
Jenny Hogan and Andrea Trevett
Animal Delivery and Care:
Alison Kriegel
Laboratory Support Services: Jennifer Labecki
Phenotyping Component
Component Director - Allen W. Cowley, Jr, Ph.D.
Co-investigators:
•Cardiac Studies:
John Baker, M.D.
•Lung Studies:
Christopher Dawson, Ph.D.
•Biochemistry Studies:
Kirkwood Pritchard, Ph.D.
•Respiratory Studies:
Hubert Forster, Ph.D.
•Isolated Vessel Studies:
Julian Lombard, Ph.D.
•Vascular and Renal Studies:
David Mattson, Ph.D.
Richard Roman, Ph.D
2 weeks
Mon. Tues. Wed. Thur. Fri.
Genomics
Component
22 rats low salt
Respiratory Studies:
hypercapnia & hypoxia
Mon. Tues. Wed. Thur. Fri.
Respiratory Studies:
response to exercise
22 rats/strain
Respiratory
Studies
Cath Surgery (next group) n=20
Animal
Production
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats high salt
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats/strain
Renal/Blood
Pressure
Studies
Cath Surgery (this group) n=20
132
rats
Mon. Tues. Wed. Thur. Fri.
Conditioning
Facility
normoxia
& hypoxia,
high salt and
low salt
22 rats high salt
22 rats low salt
Isolated Vessels: 0.4% salt;
vessels from 22 rats
Mon. Tues. Wed. Thur. Fri.
Isolated Vessels: 4% salt;
vessels from 22 rats
22 rats/strain x 2 treatments = 44 rats/strain
Isolated
Vessel
Vascular
Studies
Collect blood and tissue
22 hypoxic rats
22 normoxic rats
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Normoxia
Cardiac Studies
22 rats/strain x 2 treatments = 44 rats/strain
Mon. Tues. Wed. Thur. Fri.
22 hypoxic rats
22 normoxic rats
Airway Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Airway Studies: Normoxia
22 rats/strain x 2 treatments = 44 rats/strain
Collect blood and tissue
Collect blood and tissue
Lung Studies
Frequency (Breaths/minute)
Augmented Breaths/Minute
140
120
100
80
60
40
20
0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Frequency of Non-Augmented Breaths
in Room Air (Eupnea)
P = 0.223
BN
SS
FHH
Frequency of Augmented Breath
in Room Air (Eupnea)
P < 0.05
#
BN
SS
FHH
36
34
mm Hg
32
30
PaCO2 Control
PaCO2 Hypoxia
28
26
24
22
20
SD
SS
FHH
SSBN16
Treadmill Protocol (MAP)
Continuous measurement of MAP & HR:
Analyze Data from 30sec. before blood
draws.
Rectal
Temp
Walk
Rest
5 minutes
5 minutes
400l
Blood
draw @
(3rd min.)
Run
5 minutes
400l
Blood
draw @
(3rd min.)
Rectal
Temp
400l
Blood
draw @
(3rd min.)
2 weeks
Mon. Tues. Wed. Thur. Fri.
Genomics
Component
22 rats low salt
Respiratory Studies:
hypercapnia & hypoxia
Mon. Tues. Wed. Thur. Fri.
Respiratory Studies:
response to exercise
22 rats/strain
Respiratory
Studies
Cath Surgery (next group) n=20
Animal
Production
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats high salt
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats/strain
Renal/Blood
Pressure
Studies
Cath Surgery (this group) n=20
132
rats
Mon. Tues. Wed. Thur. Fri.
Conditioning
Facility
normoxia
& hypoxia,
high salt and
low salt
22 rats high salt
22 rats low salt
Isolated Vessels: 0.4% salt;
vessels from 22 rats
Mon. Tues. Wed. Thur. Fri.
Isolated Vessels: 4% salt;
vessels from 22 rats
22 rats/strain x 2 treatments = 44 rats/strain
Isolated
Vessel
Vascular
Studies
Collect blood and tissue
22 hypoxic rats
22 normoxic rats
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Normoxia
Cardiac Studies
22 rats/strain x 2 treatments = 44 rats/strain
Mon. Tues. Wed. Thur. Fri.
22 hypoxic rats
22 normoxic rats
Airway Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Airway Studies: Normoxia
22 rats/strain x 2 treatments = 44 rats/strain
Collect blood and tissue
Collect blood and tissue
Lung Studies
Rat Strain =
Wks of experiments =
VASCULAR PHENOTYPES
Rat 1
HIGH SALT 4.0% (HS):
HS-F
Constrictor response to Phenylephrine EC50
Constrictor response to Phenylephrine Log EC50
Dilator response to Acetycholine EC50
Dilator response to Acetycholine Log EC50
Dilator response to sodium nitroprusside EC50
Dilator response to sodium nitroprusside Log EC50
% Maximum Relaxation Acetylcholine
% Maximum Relaxation Sodium Nitroprusside
% Maximum Relaxation of Phenylephrine-Induced Contraction by 10% O2.
% Maximum Relaxation of Phenylephrine-Induced Contraction by 5% O2.
% Maximum Relaxation of Phenylephrine-Induced Contraction by 0% O2.
Fast slope of Phenylephrine-induced contraction
Slow slope of Phenylephrine-induced contraction
Rat 2
HS-F
Rat 3
HS-F
Rat 4
HS-F
Phenylephrine Response
8.0
50 )
7.5
pD 2 (-log EC
*
7.0
*
*
LowSalt
High Salt
*
* -p<0.05 vs. Low Salt
6.5
6.0
5.5
5.0
SD-F
SD-M
Dahl F
Dahl M
2 weeks
Mon. Tues. Wed. Thur. Fri.
Genomics
Component
22 rats low salt
Respiratory Studies:
hypercapnia & hypoxia
Mon. Tues. Wed. Thur. Fri.
Respiratory Studies:
response to exercise
22 rats/strain
Respiratory
Studies
Cath Surgery (next group) n=20
Animal
Production
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats high salt
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats/strain
Renal/Blood
Pressure
Studies
Cath Surgery (this group) n=20
132
rats
Mon. Tues. Wed. Thur. Fri.
Conditioning
Facility
normoxia
& hypoxia,
high salt and
low salt
22 rats high salt
22 rats low salt
Isolated Vessels: 0.4% salt;
vessels from 22 rats
Mon. Tues. Wed. Thur. Fri.
Isolated Vessels: 4% salt;
vessels from 22 rats
22 rats/strain x 2 treatments = 44 rats/strain
Isolated
Vessel
Vascular
Studies
Collect blood and tissue
22 hypoxic rats
22 normoxic rats
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Normoxia
Cardiac Studies
22 rats/strain x 2 treatments = 44 rats/strain
Mon. Tues. Wed. Thur. Fri.
22 hypoxic rats
22 normoxic rats
Airway Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Airway Studies: Normoxia
22 rats/strain x 2 treatments = 44 rats/strain
Collect blood and tissue
Collect blood and tissue
Lung Studies
Mean arterial pressure in SS and BN parental
rats and consomic chr. 13 and 16 on high salt (4.0%)
Mean Arterial Pressure
(mmHg)
180
160
140
120
100
80
SS
SS.BN13 SS.BN16
Strain
BN
PRA (ng/ml/hr)
14
High Salt
12
Low Salt
10
8
6
4
2
0
SS
BN
FHH
SSBN13
SD
30
25
SS
(females)
Change in MAP (mmHg)
Change in MAP (mmHg)
35
SSBN16
(females)
20
15
10
5
35
30
5ng
10ng
25ng
Angiotensin II (ng/kg/ml)
50ng
SSBN16
(females)
25
20
15
10
5
0
-5
0
SS (females)
0.1
0.2
0.5
1.0
Norepinephrine (µg/kg/min)
2 weeks
Mon. Tues. Wed. Thur. Fri.
Genomics
Component
22 rats low salt
Respiratory Studies:
hypercapnia & hypoxia
Mon. Tues. Wed. Thur. Fri.
Respiratory Studies:
response to exercise
22 rats/strain
Respiratory
Studies
Cath Surgery (next group) n=20
Animal
Production
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats high salt
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats/strain
Renal/Blood
Pressure
Studies
Cath Surgery (this group) n=20
132
rats
Mon. Tues. Wed. Thur. Fri.
Conditioning
Facility
normoxia
& hypoxia,
high salt and
low salt
22 rats high salt
22 rats low salt
Isolated Vessels: 0.4% salt;
vessels from 22 rats
Mon. Tues. Wed. Thur. Fri.
Isolated Vessels: 4% salt;
vessels from 22 rats
22 rats/strain x 2 treatments = 44 rats/strain
Isolated
Vessel
Vascular
Studies
Collect blood and tissue
22 hypoxic rats
22 normoxic rats
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Normoxia
Cardiac Studies
22 rats/strain x 2 treatments = 44 rats/strain
Mon. Tues. Wed. Thur. Fri.
22 hypoxic rats
22 normoxic rats
Airway Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Airway Studies: Normoxia
22 rats/strain x 2 treatments = 44 rats/strain
Collect blood and tissue
Collect blood and tissue
Lung Studies
Cardiac Conditioning and Phenotyping
Protocol
8 week
old rats
Conditioning Protocol
(16-21 Days)
Anesthesia
and Heart
Excision
30 min
perfusion
Pre-Ischemic
25 min Global
Ischemia
40 min
Reperfusion
Post-Ischemic
2 hours, 20 min
Reperfusion
Post ischemic
infarct
(%)
Post-Ischemic
Infarct
Sizesize
(% LV)
30
SS
25
SD
Hypoxic Male
20
SSBN16
15
10
FHH
5
FH
H
16
N
SS
B
SS
SD
0
Recovery developed
ventricular pressure (%)
50
45
40
35
30
Normoxic Female
25
20
15
10
5
0
SD
SS
N
B
SS
16
FH
H
2 weeks
Mon. Tues. Wed. Thur. Fri.
Genomics
Component
22 rats low salt
Respiratory Studies:
hypercapnia & hypoxia
Mon. Tues. Wed. Thur. Fri.
Respiratory Studies:
response to exercise
22 rats/strain
Respiratory
Studies
Cath Surgery (next group) n=20
Animal
Production
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats high salt
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats/strain
Renal/Blood
Pressure
Studies
Cath Surgery (this group) n=20
132
rats
Mon. Tues. Wed. Thur. Fri.
Conditioning
Facility
normoxia
& hypoxia,
high salt and
low salt
22 rats high salt
22 rats low salt
Isolated Vessels: 0.4% salt;
vessels from 22 rats
Mon. Tues. Wed. Thur. Fri.
Isolated Vessels: 4% salt;
vessels from 22 rats
22 rats/strain x 2 treatments = 44 rats/strain
Isolated
Vessel
Vascular
Studies
Collect blood and tissue
22 hypoxic rats
22 normoxic rats
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Normoxia
Cardiac Studies
22 rats/strain x 2 treatments = 44 rats/strain
Mon. Tues. Wed. Thur. Fri.
22 hypoxic rats
22 normoxic rats
Airway Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Airway Studies: Normoxia
22 rats/strain x 2 treatments = 44 rats/strain
Collect blood and tissue
Collect blood and tissue
Lung Studies
mmHg x min x kg x ml-1
Changes in Resistance at Flow =
100ml/min/g
0.25
Normoxia (F)
Hypoxia (F)
0.2
0.15
0.1
0.05
0
SS
FHH
2 weeks
Mon. Tues. Wed. Thur. Fri.
Genomics
Component
22 rats low salt
Respiratory Studies:
hypercapnia & hypoxia
Mon. Tues. Wed. Thur. Fri.
Respiratory Studies:
response to exercise
22 rats/strain
Respiratory
Studies
Cath Surgery (next group) n=20
Animal
Production
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats high salt
Mon. Tues. Wed. Thur. Fri.
Renal/BP Studies 4% salt
22 rats/strain
Renal/Blood
Pressure
Studies
Cath Surgery (this group) n=20
132
rats
Mon. Tues. Wed. Thur. Fri.
Conditioning
Facility
normoxia
& hypoxia,
high salt and
low salt
22 rats high salt
22 rats low salt
Isolated Vessels: 0.4% salt;
vessels from 22 rats
Mon. Tues. Wed. Thur. Fri.
Isolated Vessels: 4% salt;
vessels from 22 rats
22 rats/strain x 2 treatments = 44 rats/strain
Isolated
Vessel
Vascular
Studies
Collect blood and tissue
22 hypoxic rats
22 normoxic rats
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Cardiac Studies: Normoxia
Cardiac Studies
22 rats/strain x 2 treatments = 44 rats/strain
Mon. Tues. Wed. Thur. Fri.
22 hypoxic rats
22 normoxic rats
Airway Studies: Hypoxia
Mon. Tues. Wed. Thur. Fri.
Airway Studies: Normoxia
22 rats/strain x 2 treatments = 44 rats/strain
Collect blood and tissue
Collect blood and tissue
Lung Studies
Biochemical Measurements
blood draw from carotid catheter in anesthetized rats following
chronic exposure to hypoxia or normoxia
• ANP-15 (Marshfield Clinic)
Small Animal Profile (ANP-1)
• Glucose, AST, ALT, Alkaline Phosphatase, Total Bilirubin, Total
Cholesterol, Total Protein, Albumin, Urea Nitrogen, Creatinine,
Phosphorus, Calcium, Sodium, Potassium, Chloride, Bicarbonate,
Anion Gap
Complete Blood Count With Differential (CBC)
• RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, RDW,
Platelet, WBC and Differential
Genomics Component
Genotyping & Animal Production
(Transponders and transfer)
2,640
15 strains/yr
Phenotyping Component
Conditioning Protocol
Respiratory
Studies
high salt & salt depletion
Systemic Vascular
& Renal Studies
hypoxia
normoxia
Lung Studies
hypoxia
normoxia
Cardiac Studies
low salt
Isolated Vessel
Blood Studies
Genomics
Component
Research Services Component
Bioinformatics
Consomic Rat
Strains
Rat
distribution
Functional
Mapping
Component
Baseline & Stressed
Physiological Data
high salt
Baseline & Stressed
Expression Data
Expression
Profiling
TIGR Consomic
Expression Data
DNA & Genotyping
Data
Airway
R@Flow = 100ml/min/g
Resistance at 100 ml/min/gm
-1
(mmHg x min x kg x ml
0.25
0.2
0.15
Normoxia (F)
Normoxia (M)
Hypoxia (F)
Hypoxia (M)
0.1
0.05
0
SS
FHH
PGA Respiratory Protocol Timeline
•Measuring changes in Pulmonary Ventilation (VE), Tidal Volume (VT) and
frequency (f) during Hypoxia (12% O2) and Hypercapnia (7% CO2).
•Measuring Mean Arterial Pressure and Blood Gas Responses to Exercise
Monday
Tuesday
Wednesday
Week 1
Pre-surgical
Conditioning
Surgery
Week 2
Surgery &
Adaptation
Week 3
Studies
Catheter
Implantation
in Femoral
Artery
Hypoxia
Study
Treadmill
Adaptation
& Data
Analysis
Treadmill
& Box
Adaptation
Hypercapnia
Study.
Thursday
Friday
Treadmill
Adaptation 11 week old
rats
Treadmill
Adaptation
Treadmill
& Box
Adaptation
with CO2
Treadmill
& Box
Adaptation
with N2
Treadmill
Studies
Treadmill
Treadmill
Studies
Studies
Vascular Conditioning and Contraction
Protocol
Conditioning
Protocol
(16-21 days)
11 week
old rats
Pre-Surgical
Anesthesia and
Post Surgical
PhenylephrineInitial PreHypoxic
Preparations of Surgical Removal of Preparation of
induced
Conditioning
Relaxation
Aorta
Aorta
Aorta
Contractions
NaCl 4%
NaCl .4%
8:00am
10:00am
PE 10-9 to 10-2
12:00pm
Pe 10-6
10, 5, 0%
1:30-4:00pm
Vascular Conditioning and Relaxation
Protocol
Conditioning Protocol
(16-21 Days)
11
week
old
rats
0.4% or 4% NaCl
Pre-Surgical
Preparation
Anesthesia
and Aorta
Excision
Post-Surgical Initial Pre-Conditioning
Preparation
Procedures
Acetylcholine
Relaxation
1 nM to 30 uM
12:00 pm
Sodium Nitroprusside
Relaxation
0.1 nM to 30 uM
Renal Cardiovascular Phenotyping Protocol
Week 1
13
Week
old
Rats
Conditioning
Protocol (16-21
Days)
Surgical Preparation
Monday Wk 1
Surgical
Preparation
Base Line
Pressure
Recordings and
urine Collection
setup
Tuesday Wk 1 Wednesday Wk 1
Base Line
Pressure
Recordings,
Angiotensin II
Dose Response
Infusion, and
Urine collection
Thursday Wk 1
Base Line Pressure
Recordings and
Norepinephrine Dose
response Infusion
Friday Wk 1
Week 2
Base Line Recordings
for High Salt
Monday Wk 2
Base Line
Recordings
for High Salt
and Urine
collection
setup
Tuesday Wk 2
Base Line
Recordings for
High Salt Blood
collection and
La six Injection
Wednesday Wk 2
Low Salt Urine
collection setup
Thursday Wk 2
Base Line Pressure
Recordings, Urine
collection, and Blood
collection
Friday Wk 2
Lung Prep Timeline
Attach Trachea &
Place Rat in
Plethysmograph &
Insert Esophageal
Balloon
Anesthetize Rat
10
Week
old
rats
Carotid catheter
& Endotrachael Tube
Insertion
Pulmonary Artery to
Perfusion Apparatus
Remove Rat
to Surgery
Methacholine
Challenge
Heart & Lung
Extraction
~25 minutes
Marshfield
Blood
Samples via
catheter
P/Q Curve, MB+
& FAPGG
Experiments
~35 minutes
Collect Data:
•Resistance
•Compression
•Tidal Volume
•Breathing
Frequency
•Minute
Ventilation
•Extract Lungs
for Perfusion
•Extract Heart
for RV & LV
Weights
•Liver Specimen
for DNA
Processing
Collect Data:
Change in
Pressure w/ flow
FAPGG
Absorbance
MB+
Absorbance
Lung Wet
Weight
Anesthetize
Next Rat
PhysGen PGA Phenotyping Production Schedule
(Color indicates phenotyping, black indicates conditioning, * indicates additional group)
Date Wk 1
Vascular
Vascular
Lung
Lung
(Monday)
Renal
Respiratory
High Salt
Low Salt
Hypoxia
Normoxia
Cardiac
Hypoxia
Cardiac
Normoxia
4/2/2001 SSBN16, SD* SS.BN16
FHH,SSBN16 SSBN16
SD*
SSBN16,SD* SD*, 16*
SSBN16,16*
4/9/2001
FHH,SSBN16 SSBN16
SD*, 16
SD*, 16*
SD*, 16*
SSBN16*
16*
SD*, 16*
SSBN16*
4/16/2001
SSBN13
SSBN16, 13
SSBN16, 13 SD*, 16
4/23/2001 SSBN13
4/30/2001 SSBN13
SSBN13
SSBN13
SSBN16, 13
SS.BN 13
SSBN16, 13 SD*, 16, 13 16*, 13
SSBN16, 13 SBN16, 13 16*, 13
16*
SSBN16*
SSBN16*
5/7/2001 SSBN13
SSBN13
SS.BN 13
SS.BN 13
SSBN 13
16*, 13
SS.BN13
SS.BN13
SSBN13
SSBN13
SSBN13
SS.BN13
SSBN13
SSBN13
SS.BN13
SSBN13
SSBN13
SSBN13
BN
SSBN13, BN
5/14/2001 13, BN, 16*
SSBN13, BN
5/21/2001 BN, 16*
BN
5/28/2001 BN, 16*
BN
BN
BN
6/4/2001 BN, 16*
BN
BN
BN
BN
BN
BN, SSBN18
BN, SSBN18
SS.BN18
SSBN18
BN,SSBN18
BN,SSBN18
BN,SSBN18
SSBN18
BN
BN
BN
BN,SSBN18 BN
BN
BN,SSBN18 BN, SSBN18 BN, SSBN18
SS.BN18
BN, SSBN18 SS.BN18
BN
BN
BN, SSBN18
BN, SSBN18
SSBN18
SS.BN18
SS.BN18
SS.BN18
SS.BN18
SS.BN18
SS.BN18
SS.BN18
SS.BN20
SS.BN20
SS.BN20
SS.BN20
SS.BN20
SS.BN20
SS.BN20
SS.BN20
SS.BN20
SSBN20, 11 SSBN20, 11 SSBN20, 11
SSBN20, 11
SS.BN11
SSBN20, 11
SS.BN9
SS.BN9
SS.BN9
6/11/2001
6/18/2001
6/25/2001
7/2/2001
BN, 16*
SSBN18
SS.BN18
SS.BN18
7/9/2001 SS.BN18
7/16/2001 SS.BN18
7/23/2001
7/30/2001
8/6/2001
8/13/2001
8/20/2001
SS.BN20
8/27/2001
SS.BN20
9/3/2001
SS.BN20
9/10/2001 SSBN20, 11
9/17/2001 SSBN20, 11
9/24/2001
SS.BN9
10/1/2001
10/8/2001
10/15/2001
SS.BN12
10/22/2001
10/29/2001
11/5/2001
SS.BNx
11/12/2001
11/19/2001
SS.BNx
11/26/2001
12/3/2001
12/10/2001
12/17/2001
SS.BNx
12/24/2001
12/31/2001
1/7/2002
SS.BNx
SS.BN18
SS.BN18
SS.BN18
SS.BN18
SS.BN18
SS.BN20
SS.BN20
SS.BN20
SSBN20, 11
SSBN20, 11
SS.BN9
SS.BN20
SS.BN20
SS.BN20
SSBN20, 11
SS.BN11
SS.BN9
SS.BN20
SS.BN20
SS.BN20
SSBN20, 11
SSBN20, 11
SS.BN9
SS.BN20
SS.BN20
SS.BN20
SSBN20, 11
SS.BN11
SS.BN9
SS.BN12
SS.BN12
SS.BN12
SS.BN12
BN
SS.BN12
SS.BN18
SS.BN18
SS.BN12
SS.BN12
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
SS.BNx
Hypoxia & Hypercapnia Protocol
(Ventilation and MAP)
Calibration
(30 Sec.) Inject gas
Put rat in
box 20
Min.
1st Rectal Temp
Control
5”
N2 or CO2 Analyze Data
(H1)
1”
Blood
draw 400l
(Min. 3)
min. 2 & 3
Calibration
(30 Sec.)
Equil.
Analyze Data
(H2)
min. 4,5 & 6 min. 7,8 & 9
Stop Recording
2nd Rectal Temp
Blood
draw 400l
(Min 8)
Rats
Cardiac
Hypoxic
Cardiac
Normoxic
Lung
Hypoxic
Lung
Vascular
Vascular
Normoxic High Salt Low Salt
Renal
Respiratory
Bioc
hem
Assi Com
Assi Com Assi Com
Assi Com Assi Com
Assi Com Assi Com
Wea gne plet Stat Assi Com Stat gne plet Stat gne plet Stat gne plet Stat gne plet Stat gne plet Sta gne plet Stat Stat
Born ned d e us gned plete us d e us d e us d e us d e us d e tus d e us us
SD
100 100 100 75 50 100 70 50 100 0 0 100 0 0 100 100 50 100 100 50 100 70 50 100 65 25 50
SS
100 100 100 70 25 100 85 25 100 80 25 100 90 25 100 100 25 100 100 25 100 79 25 100 73 25 25
FHH
100 100 100 80 25 100 95 25 100 75 25 100 80 25 95 100 25 100
100 90 25 100 83 25 25
SSBN16 100 100 100 95 25 100
100
100
100
100
100
100
SSBN13 75 63
BN
38 25
SS.BN18April
SS.BN20June
SS.BN11July
SS.BN9 July
SS.BN12Aug