P ROGRAMS FOR G ENOMIC A PPLICATIONS

PROGRAMS FORGENOMICAPPLICATIONSNational Heart, Lung, and Blood InstitutesNational Institutes of Health

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

PGA MissionProvide 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 StructureCoordinating CommitteeBioinformaticsSubcommitteeProteomicsSubcommitteeData SharingSubcommitteeEducationSubcommitteeIntegrationSubcommitteeMicroarraySubcommitteePhenotypeSubcommittee

PGA ProgramsApplied Genomics in CardioPulmonary DiseaseJohns Hopkins University School of Medicine

Genomics of Cardiovascular Development, Adaptation, & RemodelingHarvard Medical School

Physiogenomics of Stressors in Derived Consomic RatsMedical College of Wisconsin

Genomics of Proteomics of Cell Injury and InflammationUniversity of Texas S.W. Medical Center

Innate Immunity in Heart, Lung, and Blood DiseasesThe University of Arizona

UW-FHCRC Variation Discovery ResourceUniversity of Washington

Mouse Models of Heart, Lung, and Blood DiseasesThe Jackson Laboratory

Expression Profiling of Rodent Models of Human DiseaseThe Institute for Genomics Research

Comparative Genomic Analysis of Cardiovascular GenesLawrence Berkeley National Laboratory

Genomic Analysis of Stress and InflammationHarvard Medical School

NHLBI Bay Area Functional Genomic ConsortiumThe David J. Gladstone Institute

SubcommitteeChairsBioinformaticsCarol 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/IntegrationEdward Rubin, M.D., Ph.D., The Lawrence Berkeley National Laboratory

MicroarrayJohn Quackenbush, Ph.D., The Institute for Genomics Research

PhenotypeAndrew Greene, Ph.D., Medical College of Wisconsin

ProteomicsThomas Kodadek, Ph.D., Univ. Texas S.W. Medical Center

Anticipated PGA Resources/ToolsMouse 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)

BayGenomicsApply 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. http://baygenomics.ucsf.eduPI: Dr. Stephen G. YoungFocus: Cardiopulmonary Development and Disease

CardioGenomicsTo link genes to function, dysfunction, and structural abnormalities of the cardiovascular system caused by clinically relevant genetic and environmental stimuli.

http://www.cardiogenomics.orgPI: Dr. Seigo IzumoFocus:Cardiovascular Development, Adaptation, andRemodeling

HopGenesTo 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.http://www.hopkins-genomics.org PI: Dr. Joe G.N. GarciaFocus: Tissue Remodeling in Cardiopulmonary Disease

Innate ImmunityExplore genetic susceptibility in asthma, chronic obstructive pulmonary disease, myocardial infarction and deep venous thrombosis by evaluating polymorphisms in genes involved in innate immune responses.

http://innateimmunity.netPI: Dr. Fernando D. MartinezFocus: Genetics of HLB Disorders

JAX PGAApply 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. http://pga.jax.orgPI: Dr. Luanne L. PetersFocus: Mouse Models of HLBS Disorders

PARABIOSYSTo identify and characterize the gene networks activated by pro-inflammatory, metabolic, and pathogenic stresses affecting cardiovascular and pulmonary systems. http://genetics.mgh.harvard.edu/Parabiosys/PI: Dr. Brian SeedFocus: Genetics of Inflammation and Stress

Berkeley PGAApply comparative genomics to identify and understand the role of cis-acting regulatory elements that affect the expression of cardiovascular genes. http://pga.lbl.govPI: Dr. Edward M. RubinFocus: Cardiovascular Gene Expression

PhysGenDissect multigenic common HLBS diseases through the development of panels of chromosomal substitution strains of rats (consomic rat panels).

http://pga.mcw.eduPI: Dr. Howard J. JacobsFocus: Rat Models of HLBS Disorders

Seattle SNPsTo 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. http://pga.mbt.washington.eduPI: Dr. Deborah A. NickersonFocus: Inflammation and Genetic Variability

SouthwesternElucidate the basic mechanisms underlying cell injury and inflammation through a combination of genomic and proteomic approaches. http://pga.swmed.eduPI: Dr. Stephen A. JohnstonFocus: Cell Injury and Inflammation

TREX

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. http://pga.tigr.orgPI: Dr. John QuackenbushFocus: Gene Expression in HLBS Disorders

NHLBI PGA Research Network..........HopGenes - BaltimoreCardioGenomics - BostonPhysGen - MilwaukeeSouthwestern - DallasInnateImmunity- TucsonSeattleSNPs - SeattleJAX PGA - Bar HarborTREX - RockvilleBerkeley PGA- BerkeleyParaBioSys - BostonBayGenomics- San Francisco

NHLBI PGA Web Siteshttp://www.nhlbi.nih.gov/resources/pga/index.htmPROGRAMS FOR GENOMIC APPLICATIONS

PGA Web Sites (cont.)BayGenomics - http://baygenomics.ucsf.eduCardioGenomics - http://www.cardiogenomics.orgParaBioSys - http://genetics.mgh.harvard.edu/Parabiosys/JAX PGA - http://pga.jax.orgHopGenes - http://www.hopkins-genomics.orgInnateImmunity - http://innateimmunity.net

PGA Web Sites (cont.)Berkeley PGA - http://pga.lbl.govTREX - http://pga.tigr.orgPhysGen - http://pga.mcw.eduSouthwestern - http://pga.swmed.eduSeattleSNPs - http://pga.mbt.washington.edu

NHGRI/NHLBI

Howard JacobMCWMarco MarraUBCShaying ZhaoTIGRPeter TonellatoRat GDBCHOPieter de JongUniv UtahRobert WeissRAT GENOME SEQUENCING PROJECT

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)All in MOU (with data release policy)

Time

BCM

Celera

GTC

Total

Q1

0.25/0.13

0.28/0

0.07/0.21

0.53

Q2

0.59

0.56

0.14

1.15

Q3

0.98

0.84

0.21

1.82

Q4

1.36

1.12

0.28

2.48

Strategy: Strain Selection:Howard Jacobs BN/SsNHsd

This Brown Norway strain has onlyseven heterozygous markers in themore than 4,000 tested i.e. it is very`isogenic` as a representative strain.

New BAC LibraryMade by Pieter de Jong from BN/SsNHsd female,

One enzyme so far~200 kb inserts10 X coverageSecond 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 SkimsBAC skims to cover 90% of genomeOriginally 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 Reads2kb paired end (dye term) +++10kb paired end (dye term) +++5kb and 50kb discussed,M13 (with primer chemistry) ?

- Usual- Assembly- Data `purity`Challenges:

Atlas:

A whole genome assembler

Lung StudieshypoxianormoxiaIsolated VesselResearch Services ComponentPhenotyping ComponentConditioning ProtocolGenomicsComponentProfilingRespiratoryStudiesBioinformatics ComponentConsomic Rat StrainsFunctionalMappingDNA & GenotypingDataBaseline & StressedExpression DataRatdistributionBaseline & StressedPhysiological DataGenomics ComponentGenotyping & Animal Production(Transponders and transfer)TIGR ConsomicExpression Data Expression low salthigh saltBlood Studies2,64015 strains/yr

330330330330330330330330

Mary Pat Kunert, RN, Ph.D., Project SupervisorMindy Dwinell, Ph.D. Project SupervisorMeredith Skelton, M.S. , Sr. Research AssociateJulie Messinger, M.S., Program Coordinator

Cardiac Studies:Jessica Laessig and William HutchinsRenal Studies:Larry Clowry and Mike Bregantini Biochemistry:Candace JonesLung Studies:Jess Powlas and Bernadette CabigasIsolated Vessel Studies:Janelle Yarina Respiratory Studies:Jenny Hogan and Andrea TrevettAnimal Delivery and Care: Alison KriegelLaboratory Support Services:Jennifer LabeckiPhenotyping Team

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.DComponent Director - Allen W. Cowley, Jr, Ph.D.Phenotyping Component

P = 0.223

Treadmill Protocol (MAP)RestWalkRun5 minutes5 minutes5 minutes400l Blood draw @(3rd min.)400l Blood draw @(3rd min.)400l Blood draw @(3rd min.)Rectal

TempRectal

TempContinuous measurement of MAP & HR: Analyze Data from 30sec. before blood draws.

vascular

Rat Strain =HS-F

Wks of experiments =HS-M

VASCULAR PHENOTYPESRat 1Rat 2Rat 3Rat 4RAT 5RAT 6RAT 7RAT 8RAT 9RAT 10RAT 11RAT 12RAT 13RAT 14RAT 15RAT 16RAT 17RAT 18RAT 19RAT 20RAT 21RAT 22LS-F

HIGH SALT 4.0% (HS):HS-FHS-FHS-FHS-FHS-FHS-FHS-FHS-FHS-FHS-FHS-MHS-MHS-MHS-MHS-MHS-MHS-MHS-MHS-MHS-MLS-F/PSLS-M/PSLS-M

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.



Fast slope of Phenylephrine-induced contraction

Slow slope of Phenylephrine-induced contraction

Maximum force (g) per wet weight of aorta (contraction)

Maximum force (g) per wet weight of aorta (relaxation)

RAT 23RAT 24RAT 25RAT 26RAT 27RAT 28RAT 29RAT 30RAT 31RAT 32RAT 33RAT 34RAT 35RAT 36RAT 37RAT 38RAT 39RAT 40RAT 41RAT 42RAT 43RAT 44

LOW SALT 0.4% (LS):LS-FLS-FLS-FLS-FLS-FLS-FLS-FLS-FLS-FLS-FLS-MLS-MLS-MLS-MLS-MLS-MLS-MLS-MLS-MLS-MLS-F/PSLS-M/PS








% Maximim Relaxation Sodium Nitroprusside




Fast slope of Phenylephrine-induced contraction

Slow slope of Phenylephrine-induced contraction

Maximum force (g) per wet weight of aorta (contraction)

Maximum force (g) per wet weight of aorta (relaxation)

&C&"Arial,Bold Italic"&16VASCULARPGA

Vascular.stats

Rat strain =

Wks of experiments =

VASCULAR PHENOTYPES

HIGH SALT 4.0% (HS):Mean (HS-T)Std Dev (HS-T)Std Error (HS-T)Max(HS-T)Min(HS-T)Mean (HS-F)Std Dev (HS-F)Std Error (HS-F)Max (HS-F)Min (HS-F)Mean (HS-M)Std Dev (HS-M)Std error (HS-M)Max (HS-M)Min (HS-M)

Constrictor response to Phenylephrine EC500.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Constrictor response to Phenylephrine Log EC500.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Dilator response to Acetycholine EC500.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Dilator response to Acetycholine Log EC500.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Dilator response to sodium nitroprusside EC500.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Dilator response to sodium nitroprusside Log EC500.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

% Maximum Relaxation Acetylcholine0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

% Maximum Relaxation Sodium Nitroprusside0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

% Maximum Relaxation of Phenylephrine-Induced Contraction by 10% O2.0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00



Fast slope of Phenylephrine-induced contraction0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Slow slope of Phenylephrine-induced contraction0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Maximum force (g) per wet weight of aorta (contraction)0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

Maximum force (g) per wet weight of aorta (relaxation)0.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+000.00E+0000.00E+000.00E+000.00E+00

LOW SALT 0.4% (LS):Mean (LS-T)Std Dev (LS-T)Std Error (LS-T)Max(LS-T)Min(LS-T)Mean (LS-F)Std Dev (LS-F)Std Error (LS-F)Max (LS-F)Min (LS-F)Mean (LS-M)Std Dev (LS-M)Std error (LS-M)Max (LS-M)Min (LS-M)







% Maximum Relaxation Acetylcholine000000000000000

% Maximim Relaxation Sodium Nitroprusside000000000000000

% Maximum Relaxation of Phenylephrine-Induced Contraction by 10% O2.000000000000000



Fast slope of Phenylephrine-induced contraction000000000000000

Slow slope of Phenylephrine-induced contraction000000000000000

Maximum force (g) per wet weight of aorta (contraction)000000000000000

Maximum force (g) per wet weight of aorta (relaxation)000000000000000

&C&"Arial,Bold"&12VascularStatistics

CX Response EC50

00

00

00

00

00

00

00

00

00

00

HS-F

HS-M

LS-F

LS-M

Constrictor Response to Phenylephrine EC50

Vascular PGA

CX Response LOG EC50

HS-F

HS-M

LS-F

LS-M

Constrictor Response to Phenylephrine Log EC50

Vascular PGA

DL Response ACH EC50

HS-F

HS-M

LS-F

LS-M

Dilator Response to Acetylcholine EC50

Vascular PGA

DL Response ACH Log EC50

HS-F

HS-M

LS-F

LS-M


Vascular PGA

DL Response SNP EC50

HS-F

HS-M

LS-F

LS-M

Dilator Response to SNP EC50

Vascular PGA

DL Response SNP Log EC50

HS-F

HS-M

LS-F

LS-M

Dilator Response to SNP Log EC50

Vascular PGA

% Maximum RX ACH

HS-F

HS-M

LS-F

LS-M


Vascular PGA

% Maximum RX SNP

HS-F

HS-M

LS-F

LS-M


Vascular PGA

% Maximum RX of PE CX by 10%O2

HS-F

HS-M

LS-F

LS-M

% Maximum Relaxation of Phenylephrine Induced Contraction by 10% O2

Vascular PGA

% Maximum RX of PE CX by 5% O2

HS-F

HS-M

LS-F

LS-M


Vascular PGA

% Maximum RX of PE CX by 0% O2

HS-F

HS-M

LS-F

LS-M


Vascular PGA

Maximum Force (CX Protocol)

HS-F

HS-M

LS-F

LS-M

Maximum Force (g)/wet weight of aorta (Contraction protocol)

Vascular PGA

Maximum Force (RX Protocol)

HS-F

HS-M

LS-F

LS-M

Maximum Force (g)/wet weight of aorta (Relaxation Protocol)

Vascular PGA

Means Phenotype #1

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0

Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)

Rat strain = Wks of experiments = VASCULAR PHENOTYPES

Mean (HS-T)

Mean (LS-T)

Constrictor Response to Phenylephrine EC50

Vascular PGA

Means Phenotype #2

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)

Constrictor Response to Phenylephrine Log EC50

Vascular PGA

Means Phenotype #3

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)


Vascular PGA

Means Phenotype #4

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)

Dilator Response to Acetylcholine LogEC50

Vascular PGA

Means Phenotype #5

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Mean (HS-F)

Mean (LS-F)

Mean (LS-M)

Mean (HS-M)


Mean (HS-T)

Mean (LS-T)

Dilator Response to Sodium Nitroprusside EC50

Vascular PGA

Means Phenotype #6

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)

Dilator Response to Sodium Nitroprusside Log EC50

Vascular PGA

Means Phenotype #7

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)


Vascular PGA

Means Phenotype #8

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)


Vascular PGA

Means Phenotype #9

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)

% Maximum Relaxation of Phenylephrine-induced Contraction to 10% O2

Vascular PGA

Means Phenotype #10

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)


Vascular PGA

Means Phenotype #11

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)


Vascular PGA

Means Phenotype #12

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)

Maximum Force (g) per wet weight of aorta (Contraction protocol)

Vascular PGA

Means Phenotype #13

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Mean (HS-F)

Mean (LS-F)

Mean (HS-M)

Mean (LS-M)


Mean (HS-T)

Mean (LS-T)

Maximum Force (g) per wet weight of aorta (Relaxation protocol)

Vascular PGA

Mean arterial pressure in SS and BN parentalrats and consomic chr. 13 and 16 on high salt (4.0%)

Chart1

2.22428571433.30214285710.50.7

1.579.3

9.311.834375

0.62.4

2.47583333335.7477777778

High Salt

Low Salt

PRA (ng/ml/hr)

SS

BN

SSBN13

FHH

SD

Sheet1

SSBNFHHSSBN13SD

SS rats1.0220.852.592.040.931.197.831.410.91.66.580.841.362.18681262360.5844502576HS PRA2.22428571430.61.570.59.30.70.62.47583333330.4

2.674.961.993.412.773.873.526.541.581.027.662.162.281.81.91730651850.5124217212

LS PRA3.30214285710.59.30.911.8343750.52.45.74777777780.8

FHH rats3.529.5512.4511.1413.039.398.328.3810.188.765.5711.8411.717.398.29.29533333332.599494090.6711864879Change is 1.8+/-0.3

10.1613.9713.213.4611.4611.748.7111.769.0813.116.112.5712.0111.9810.189.8711.8343751.9384047350.4846011837

SD rats1.961.91.750.310.8753.733.632.643.991.342.591.39131369140.4016376671

8.334.334.979.65.966.961.855.943.792.36377757930.7879258598

HS Protein excretion1.273.3400.8502.121.4801.92.42.76.782.11.901.7893333333

Sheet1

0.50.7

HS

LS

PRA (ng/ml/hr)

SD

FHH

SSBN13

BN

SS

Sheet2

Sheet3

Chart3

2.64111111118

18.913333333312

29.433333333325

31.111111111132

SS (females)

SSBN16 (females)

Angiotensin II (ng/kg/ml)

Change in MAP (mmHg)

50ng

25ng

10ng

5ng

Sheet1

SSBNFHHSSBN13SD

SS rats1.0220.852.592.040.931.197.831.410.91.66.580.841.362.18681262360.5844502576HS PRA2.22428571430.61.570.59.30.72.47583333330.4

2.674.961.993.412.773.873.526.541.581.027.662.162.281.81.91730651850.5124217212

LS PRA3.30214285710.59.30.911.8343750.55.74777777780.8


10.1613.9713.213.4611.4611.748.7111.769.0813.116.112.5712.0111.9810.189.8711.8343751.9384047350.4846011837

SD rats1.961.91.750.310.8753.733.632.643.991.342.591.39131369140.4016376671

8.334.334.979.65.966.961.855.943.792.36377757930.7879258598SD Protein excretion0.23642857140.13647243410.0364737922

BN Protein excretion19.210

SS Protein excretion1.273.3400.8502.121.4801.92.42.76.782.11.901.7893333333SS Protein excretion1.78933333331.68915152930.4222878823

SSBN13 Protein excretion5522

FHH Protein excretion13.12520.255.2516.27534.237.9523.587534.42559.62557.787531.3537.841.8520.92555.895.4SSBN16 Protein excretion

FHH Protein excretion32.6816.50852580054.1271314501

SD Protein excretion0.330.190.140.30.280.20.040.070.580.180.330.190.150.33

SSBN13 Protein excretion55+/-226.2-2.30.47161405.35.825

11142.32.5232503.510.1625

SSBN16 Protein excretion1217-4.47233201212.325

2321-2.71464901315.4125

BN Protein excretion19.2+/-10

SSBN16SDSS

5ng85.8252.6411111111

10ng1210.162518.9133333333

25ng2512.32529.4333333333

50ng3215.412531.1111111111

Sheet1

2.22428571433.30214285710.50.7

1.579.3

9.311.834375

2.47583333335.7477777778

HS

LS

PRA (ng/ml/hr)

SD

FHH

SSBN13

BN

SS

Sheet2

0.236428571419.21.78933333335532.68

Urinary Protein excretion (mg/24hrs)

SD

BN

SS

SSBN13

FHH

Sheet3

82.64111111115.825

1218.913333333310.1625

2529.433333333312.325

3231.111111111115.4125

SSBN16 (females)

SS (females)

SD (females)

Change in MAP (mmHg) to Ang II

50ng

25ng

10ng

5ng

82.64111111115.825

1218.913333333310.1625

2529.433333333312.325

3231.111111111115.4125

SSBN16 (females)

SS (females)

SD (females)


5ng

10ng

25ng

50ng

Chart4

3.6777777778-2

4.67777777781

17.3254.5

31.666666666712

SS (females)

SSBN16 (females)

Norepinephrine (g/kg/min)


1.0

0.2

0.1

0.5

Sheet1

SSBNFHHSSBN13SD

SS rats1.0220.852.592.040.931.197.831.410.91.66.580.841.362.18681262360.5844502576HS PRA2.22428571430.61.570.59.30.72.47583333330.4

2.674.961.993.412.773.873.526.541.581.027.662.162.281.81.91730651850.5124217212

LS PRA3.30214285710.59.30.911.8343750.55.74777777780.8


10.1613.9713.213.4611.4611.748.7111.769.0813.116.112.5712.0111.9810.189.8711.8343751.9384047350.4846011837

SD rats1.961.91.750.310.8753.733.632.643.991.342.591.39131369140.4016376671

8.334.334.979.65.966.961.855.943.792.36377757930.7879258598SD Protein excretion0.23642857140.13647243410.0364737922

BN Protein excretion19.210

SS Protein excretion1.273.3400.8502.121.4801.92.42.76.782.11.901.7893333333SS Protein excretion1.78933333331.68915152930.4222878823

SSBN13 Protein excretion5522

FHH Protein excretion13.12520.255.2516.27534.237.9523.587534.42559.62557.787531.3537.841.8520.92555.895.4SSBN16 Protein excretion

FHH Protein excretion32.6816.50852580054.1271314501

SD Protein excretion0.330.190.140.30.280.20.040.070.580.180.330.190.150.33

SSBN13 Protein excretion55+/-22

SSBN16 Protein excretion

BN Protein excretion19.2+/-10

SSBN16SDSS

5ng8

10ng12

25ng25

50ng32

0.1-26.421253.6777777778

0.2121.3254.6777777778

0.54.512.37517.325

11215.287531.6666666667

-3.6-4.5335-1.53.74.1-0.7-2.43.6777777778

2.51.62.12.81.67.910.25.38.14.6777777778

12216202732182.617.32517.325

36302135404334182831.6666666667

Sheet1

000.50.7

00

00

00

00

HS

LS

PRA (ng/ml/hr)

SD

FHH

SSBN13

BN

SS

Sheet2

000000

Urinary Protein excretion (mg/24hrs)

SD

BN

SS

SSBN13

FHH

Sheet3

0

0

0

0

SSBN16 (females)


50ng

25ng

10ng

5ng

000

000

000

000

SD (females)

SS (females)

SSBN16 (females)

Norepinephrine (g/kg/min)


Cardiac Conditioning and Phenotyping Protocol 8 week old rats

Sheet1

Conditioning Protocol (16-21 Days)Anesthesia and Heart Excision30 min perfusion25 min Global Ischemia40 min Reperfusion2 hours, 20 min Reperfusion

Pre-IschemicPost-Ischemic

Sheet2

Sheet3

Post ischemic infarct size (%)

Recovery developed ventricular pressure (%)

blood draw from carotid catheter in anesthetized rats following chronic exposure to hypoxia or normoxiaANP-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 DifferentialBiochemical Measurements

Lung StudieshypoxianormoxiaIsolated VesselResearch Services ComponentPhenotyping ComponentConditioning ProtocolGenomicsComponentProfilingRespiratoryStudiesBioinformatics ComponentConsomic Rat StrainsFunctionalMappingDNA & GenotypingDataBaseline & StressedExpression DataRatdistributionBaseline & StressedPhysiological DataGenomics ComponentGenotyping & Animal Production(Transponders and transfer)TIGR ConsomicExpression Data Expression low salthigh saltBlood Studies2,64015 strains/yr

330330330330330330330330

Airway Resistance at 100 ml/min/gm

PGA Respiratory Protocol TimelineMeasuring changes in Pulmonary Ventilation (VE), Tidal Volume (VT) and frequency (f) during Hypoxia (12% O2) and Hypercapnia (7% CO2).

Treadmill Studies

Treadmill Studies

Hyper-capniaStudy.Treadmill Adaptation & Data Analysis

Hypoxia Study

Treadmill& Box Adaptation with N2

Treadmill& Box Adaptation with CO2Treadmill& Box Adaptation Surgery Catheter Implantation in Femoral Artery

TreadmillAdaptationTreadmillAdaptation -11 week old rats Monday Tuesday Wednesday Thursday Friday

Week 1Pre-surgical ConditioningWeek 2Surgery &AdaptationWeek 3StudiesMeasuring Mean Arterial Pressure and Blood Gas Responses to ExerciseTreadmill Studies

11 week old ratsVascular Conditioning and Contraction Protocol

Vascular Conditioning and Relaxation Protocol 11 week old rats12:00 pm

Sheet1

Conditioning Protocol (16-21 Days)Pre-Surgical PreparationAnesthesia and Aorta ExcisionPost-Surgical PreparationInitial Pre-Conditioning ProceduresAcetylcholine RelaxationSodium Nitroprusside Relaxation

0.4% or 4% NaCl1 nM to 30 uM

Sheet2

Sheet3

Sheet1

Conditioning Protocol (16-21 Days)Pre-Surgical PreparationAnesthesia and Aorta ExcisionPost-Surgical PreparationInitial Pre-Conditioning ProceduresAchetylcholine RelaxationSodium Nitroprusside Relaxation

0.1 nM to 30 uM

Sheet2

Sheet3

Renal Cardiovascular Phenotyping Protocol

13 Weekold RatsWeek 1Week 2

Sheet1

Conditioning Protocol (16-21 Days)Surgical PreparationSurgical PreparationBase Line Pressure Recordings and urine Collection setupBase Line Pressure Recordings, Angiotensin II Dose Response Infusion, and Urine collectionBase Line Pressure Recordings and Norepinephrine Dose response Infusion

Monday Wk 1Tuesday Wk 1Wednesday Wk 1Thursday Wk 1Friday Wk 1

Sheet2

Sheet3

Sheet1

Base Line Recordings for High SaltBase Line Recordings for High Salt and Urine collection setupBase Line Recordings for High Salt Blood collection and Lasix InjectionLow Salt Urine collection setupBase Line Pressure Recordings, Urine collection, and Blood collection

Monday Wk 2Tuesday Wk 2Wednesday Wk 2Thursday Wk 2Friday Wk 2

Sheet2

Sheet3

Carotid catheter & Endotrachael TubeInsertionMethacholineChallengeHeart & Lung ExtractionP/Q Curve, MB+& FAPGG Experiments10 Week old ratsLung Prep TimelineMarshfield Blood Samples via catheter~25 minutes

Collect Data:ResistanceCompressionTidal VolumeBreathing FrequencyMinute VentilationExtract Lungs for PerfusionExtract Heart for RV & LV WeightsLiver Specimen for DNA ProcessingCollect Data:Change in Pressure w/ flowFAPGG AbsorbanceMB+ AbsorbanceLung Wet Weight~35 minutesAnesthetize RatPlace Rat in Plethysmograph & Insert Esophageal BalloonRemove Rat to SurgeryAttach Trachea & Pulmonary Artery to Perfusion ApparatusAnesthetize Next Rat

Revision 8

PhysGen PGA Phenotyping Production Schedule

(Color indicates phenotyping, black indicates conditioning, * indicates additional group)(Vascular group using nave animals; study starts wk 1 of 5 wks)

Date Wk 1 (Monday)RenalRespiratoryVascular High SaltVascular Low SaltLung HypoxiaLung NormoxiaCardiac HypoxiaCardiac NormoxiaComments

12/18/00SDSDSDSDSDSDSDRespiratory will do their SD group starting 2/5.

12/25/00SDSDSDSDSDSDSD2 weeks for hypoxia chamber modifications, repairs

1/1/01SDSDSDSDSDSDSD

1/8/01SDSD, SSSD,SSSDSDSDSDstart Vascular group for best age fit

1/15/01SD, SSSDSSSD, SSSSSD, SSSSSD, SSstart SS Cardiac, Lung, renal groups; Respiratory do SD group

1/22/01SSSDSSSSSSSSSSSS

1/29/01SSSD, SSSSSSSSSSSSSS

2/5/01SS, FHHSD, SSSS,FHHSS,FHHSS,FHHSS, FHHSS,FHHstart all groups but Respiratory and High Salt Vascular

2/12/01SS, FHHSD,SS,FHHSD*,FHHFHHSS,FHHFHHSS, FHHstart Respiratory group for best age fit; Vascular group study Low Salt SD group of females 2/12

2/19/01FHH, SS*SS,FHHSS-RenalFHHFHHFHHFHHFHH, SD*Vascular study on rats 2/19 after SS Renal study - for comparison

2/26/01FHH, SS*SS,FHHFHH, SD*FHHFHHFHHFHH, SD*Renal surgery 2/26 in 5th fl.lab

3/5/01FHH, SS*, 16FHH,SSBN16SS-Resp, SD*FHH, 16SS.BN16FHH, SD*, 16Downtime for hypoxia chamber maintenance w/o 3/5; Vascular study on 3/12 after SS Resp study

3/12/01SS*, 16FHH,SSBN16Progesterone-SD, SD*SSBN16,SD*SS.BN16SD*, SSBN16Phenotyper training 12th; Vascular group do progesterone study on SD 3/5; Renal surgery 5th fl.

3/19/01SS*, 16SS.BN16FHHSD*SSBN16,SD*SSBN16SD*, SSBN16Phenotyper training 19th

3/26/01SSBN16, SD*SS.BN16FHHSD*SSBN16,SD*SSBN16,SD*SS.BN16

4/2/01SSBN16, SD*SS.BN16FHH,SSBN16SSBN16SD*SSBN16,SD*SD*, 16*SSBN16,16*

4/9/01FHH,SSBN16SSBN16SD*, 16SD*, 16*SD*, 16*SSBN16*PGA-CC meeting in Milwaukee

4/16/01SSBN13SSBN16, 13SSBN16, 13SD*, 1616*SD*, 16*SSBN16*

4/23/01SSBN13SSBN13SSBN16, 13SSBN16, 13SD*, 16, 1316*, 1316*SSBN16*

4/30/01SSBN13SSBN13SS.BN 13SSBN16, 13SBN16, 1316*, 13SSBN16*holiday 5/28 Monday, affects week 5

5/7/01SSBN13SSBN13SS.BN 13SS.BN 13SSBN 1316*, 13SS.BN13SS.BN13holiday 5/28 Monday, affects week 4

5/14/0113, BN, 16*SSBN13, BNSSBN13SSBN13SSBN13SS.BN13SSBN13

5/21/01BN, 16*BNSSBN13SS.BN13SSBN13

5/28/01BN, 16*BNBNBNSSBN13SSBN13First data release June 1

6/4/01BN, 16*BNBNBNBNBNBNSSBN13, BNholiday 7/4 Wednesday, affects week 5

6/11/01BN, 16*BNBN, SSBN18BN,SSBN18BNBNBNBNMidwest Phys. Society meeting - Madison 6/11-12, holiday 7/4 Wednesday, affects week 4

6/18/01SSBN18BN, SSBN18BN,SSBN18BN,SSBN18BNBNBNDowntime for hypoxia chamber maintenance 6/18

6/25/01SS.BN18SS.BN18BN,SSBN18BN,SSBN18BN, SSBN18BN, SSBN18BN, SSBN18

7/2/01SS.BN18SSBN18SSBN18SS.BN18BN, SSBN18SS.BN18BN, SSBN18

7/9/01SS.BN18SS.BN18SSBN18SS.BN18SS.BN18SS.BN18SS.BN18

7/16/01SS.BN18SS.BN18SS.BN18SS.BN18SS.BN18Second data release September 7

7/23/01SS.BN18SS.BN18SS.BN18

7/30/01SS.BN18

8/6/01SS.BN18holiday 9/3 Monday, affects week 5

8/13/01holiday 9/3 Monday, affects week 4

8/20/01SS.BN20SS.BN20SS.BN20SS.BN20SS.BN20SS.BN20SS.BN20SS.BN20



9/10/01SSBN20, 11SSBN20, 11SSBN20, 11SSBN20, 11SSBN20, 11SSBN20, 11SSBN20, 11SSBN20, 11

9/17/01SSBN20, 11SSBN20, 11SS.BN11SSBN20, 11SS.BN11SSBN20, 11SS.BN11SSBN20, 11Downtime for hypoxia chamber maintenance w/o 9/17


10/1/01

10/8/01

10/15/01SS.BN12SS.BN12SS.BN12SS.BN12SS.BN12SS.BN12SS.BN12SS.BN12Third data release December 7

10/22/01holiday 11/22 & 23 Thursday & Friday, affects week 5

10/29/01holiday 11/22 & 23 Thursday & Friday, affects week 4

11/5/01SS.BNxSS.BNxSS.BNxSS.BNxSS.BNxSS.BNxSS.BNxSS.BNx

11/12/01


11/26/01holiday 12/24 & 25 Monday & Tuesday, affects week 5

12/3/01holiday 12/24 & 25 Monday, Tuesday, affects week 4, holiday 1/1 Tuesday affects week 5

12/10/01holiday 1/1 Tuesday, affects week 4, downtime for hypoxia maintenance 12/10


12/24/01

12/31/01


Total Complete Calendar year 111 consomic, 4 parental

Total Complete in Grant year 1 (9/00-8/01)3 consomic, 4 parental

summary of 5 week protocol

week 1 - high salt diet renal and vascular

week 2 - hypoxia conditioning

week 3 - hypoxia conditioning; respiratory conditioning

week 4 - renal and respiratory surgery; all groups - studies

week 5 - all groups - studies

&LJ. Messinger&CPage &P of &N&R&D

Hypoxia & Hypercapnia Protocol(Ventilation and MAP)Put rat in box 20 Min.ControlInject gas N2 or CO2 Equil.

Stop Recording1st Rectal Temp 5 1 min. 2 & 3 min. 4,5 & 6 min. 7,8 & 9 2nd Rectal TempBlood draw 400l (Min. 3)Blooddraw 400l (Min 8)Calibration (30 Sec.)Calibration (30 Sec.)Analyze Data (H1) Analyze Data (H2)

PhysGen PGA production summary

RatsCardiacCardiacLungLungVascularVascularRenalRespiratory

HypoxicNormoxicHypoxicNormoxicHigh SaltLow SaltBiochem

BornWeanedAssignedCompleteStatusAssignedCompleteStatusAssignedCompleteStatusAssignedCompleteStatusAssignedCompleteStatusAssignedCompleteStatusAssignedCompleteStatusAssignedCompleteStatusStatus

SD10010010075501007050100001000010010050100100501007050100652550

SS100100100702510085251008025100902510010025100100251007925100732525

FHH100100100802510095251007525100802595100251001009025100832525

SSBN161001001009525100100100100100100100

SSBN137563

BN3825

SS.BN18April

SS.BN20June

SS.BN11July

SS.BN9July

SS.BN12Aug

Definitions:

Assigned = percentage of # assigned out of 20 possible (or 2 if the control), at transfer to phenotyping

Complete = percentage of # that generated data from phenotyping out of # assigned

Status: 0% = data not analyzed by phenotypers

25% = data completed by phenotypers

50% = data approved by Mary Pat and PI's (may result in a change to percent complete)

75% = data packaged by RSC

100% = data on the web

&CPGA Production Summary(%)

&LJ. Messinger&R&D

Documents

P ROGRAMS FOR G ENOMIC A PPLICATIONS