PROGRAMS FOR GENOMIC APPLICATIONS

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PROGRAMS FORGENOMICAPPLICATIONS
National Heart, Lung, and Blood
Institutes National Institutes of Health
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Mission Statement

• To develop new resources, reagents, and education
  programs for investigators engaged in
  NHLBI-related research.

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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.

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Organizational Structure
Coordinating Committee
Bioinformatics Subcommittee
Phenotype Subcommittee
Proteomics Subcommittee
Microarray Subcommittee
Data Sharing Subcommittee
Integration Subcommittee
Education Subcommittee
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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 Genomics of Proteomics of
Cell Injury and Inflammation University of Texas
S.W. Medical Center Innate Immunity in Heart,
Lung, and Blood Diseases The University of
Arizona UW-FHCRC Variation Discovery
Resource University of Washington
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 Lawrence
Berkeley National Laboratory Genomic Analysis of
Stress and Inflammation Harvard Medical
School NHLBI Bay Area Functional Genomic
Consortium The David J. Gladstone Institute
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SubcommitteeChairs

• Bioinformatics
• Carol Bult, Ph.D., The Jackson Laboratory
• Data Sharing
• Isaac Kohane, M.D., Ph.D., Harvard Medical School
• 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

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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)

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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
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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
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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
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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
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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
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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
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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
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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
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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 inter-individual variation and its
  relationship to disease risk, outcome and
  treatments for common human disorders.

PI Dr. Deborah A. Nickerson
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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
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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
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NHLBI PGA Research Network

• SeattleSNPs - Seattle

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.

• PhysGen - Milwaukee

• JAX PGA - Bar Harbor

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• CardioGenomics - Boston

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• ParaBioSys - Boston

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.

• HopGenes - Baltimore

• BayGenomics
• - San Francisco

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• TREX - Rockville

• Berkeley PGA
• - Berkeley

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• InnateImmunity
• - Tucson

• Southwestern - Dallas

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NHLBI PGA Web Sites
PROGRAMS FOR GENOMIC APPLICATIONS
http//www.nhlbi.nih.gov/resources/pga/index.htm
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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/Parab
iosys/
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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
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RAT GENOME SEQUENCING PROJECT
NHGRI/NHLBI Howard Jacob MCW Marco
Marra UBC Shaying Zhao TIGR Peter Tonellato Rat
GDB CHO Pieter de Jong Univ Utah Robert Weiss
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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,
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Fold-Coverage Timelines (Year One) (Cumulative)
All in MOU (with data release policy)
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Strategy Strain Selection
Howard Jacobs 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.
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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,
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Fingerprints UBC, 40,000 for
de-randomization 200,000 for fprint
assembly, BES TIGR, 200,000 paired ends
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Strategy BAC Skims
BAC skims to cover 90 of genome Originally
20,000 150 kb de-randomized BACs _at_ 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 _at_768 reads per BAC
and 1.7 X coverage
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Strategy BAC Skims (contd)
Reads in BACs 2kb paired end (dye term)
10kb paired end (dye term) -- M13 (with
primer chemistry) ?
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Strategy WGS Reads
2kb paired end (dye term) 10kb paired end
(dye term) 5kb and 50kb discussed, M13 (with
primer chemistry) ?
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Challenges
- Usual - Assembly - Data purity
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Atlas A whole genome assembler
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Genomics Component
Genotyping Animal Production
(Transponders and transfer)
2,640 15 strains/yr
Phenotyping
Component
Conditioning Protocol
330
330
330
330
330
330
330
330
high salt
low salt
hypoxia
normoxia
Respiratory
Lung Studies
Isolated Vessel
Studies
Blood Studies
Genomics
Component
Expression
Research Services Component
Profiling
Bioinformatics
Component
TIGR
Consomic
Expression Data
Consomic
Rat
Functional
Strains
Baseline Stressed
Baseline Stressed
DNA
Genotyping
Mapping
Expression Data
Physiological Data
Data
Rat
distribution
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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
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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

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P 0.223
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Treadmill Protocol (MAP)
Continuous measurement of MAP HR Analyze Data
from 30sec. before blood draws.
Rest
Walk
Run
Rectal Temp
Rectal Temp
5 minutes
5 minutes
5 minutes
400?l Blood draw _at_ (3rd min.)
400?l Blood draw _at_ (3rd min.)
400?l Blood draw _at_ (3rd min.)
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Mean arterial pressure in SS and BN parental rats
and consomic chr. 13 and 16 on high salt (4.0)
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Cardiac Conditioning and Phenotyping Protocol
8 week old rats
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Post ischemic infarct size ()
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Recovery developed ventricular pressure ()
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blood draw from carotid catheter in anesthetized
rats following chronic exposure to hypoxia or
normoxia
Biochemical Measurements

• 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

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Genomics Component
Genotyping Animal Production
(Transponders and transfer)
2,640 15 strains/yr
Phenotyping
Component
Conditioning Protocol
330
330
330
330
330
330
330
330
high salt
low salt
hypoxia
normoxia
Respiratory
Lung Studies
Isolated Vessel
Studies
Blood Studies
Genomics
Component
Expression
Research Services Component
Profiling
Bioinformatics
Component
TIGR
Consomic
Expression Data
Consomic
Rat
Functional
Strains
Baseline Stressed
Baseline Stressed
DNA
Genotyping
Mapping
Expression Data
Physiological Data
Data
Rat
distribution
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Airway Resistance at 100 ml/min/gm
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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
Thursday Friday
Treadmill Adaptation -11 week old rats
Treadmill Adaptation
Week 1 Pre-surgical Conditioning
Treadmill Box Adaptation with N2
Surgery Catheter Implantation in Femoral Artery
Treadmill Box Adaptation
Treadmill Box Adaptation with CO2
Week 2 Surgery Adaptation
Hypoxia Study
Treadmill Studies
Treadmill Studies
Hyper-capnia Study.
Treadmill Studies
Treadmill Adaptation Data Analysis
Week 3 Studies
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Vascular Conditioning and Contraction Protocol
11 week old rats
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Vascular Conditioning and Relaxation Protocol
11 week old rats
1200 pm
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Renal Cardiovascular Phenotyping Protocol
Week 1
13 Week old Rats


Week 2
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Lung Prep Timeline
Attach Trachea Pulmonary Artery to Perfusion
Apparatus
Place Rat in Plethysmograph Insert Esophageal
Balloon
Anesthetize Next Rat
Anesthetize Rat
Remove Rat to Surgery
10 Week old rats
Carotid catheter Endotrachael Tube Insertion
Methacholine Challenge
Heart Lung Extraction
P/Q Curve, MB FAPGG Experiments
25 minutes
35 minutes
Marshfield Blood Samples via catheter

• 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

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Hypoxia Hypercapnia Protocol(Ventilation and
MAP)
Calibration (30 Sec.)
Calibration (30 Sec.)
Inject gas N2 or CO2
Analyze Data (H1)
Analyze Data (H2)
Put rat in box 20 Min.
Stop Recording
Control
Equil.
1st Rectal Temp 5 1
min. 2 3 min. 4,5 6 min. 7,8 9
2nd Rectal Temp
Blood draw 400?l (Min. 3)
Blood draw 400?l (Min 8)
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