HighThroughput Phenotyping Finds New Mouse Models of Heart, Lung, Blood and Sleep Disorders: JAX PGA

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High-Throughput Phenotyping Finds New Mouse
Models of Heart, Lung, Blood and Sleep Disorders
JAX PGAKaren L. SvensonNHLBI Symposium
TutorialJuly 23-24, 2003
Major Goal To link genetic variation to
biological function and dysfunction

• Mouse
• Phenotype-Driven Approach to Gene Discovery
• Inbred Strain Characterization
• Mutagenesis

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HLB Program Components

• Strain Characterization
• Mutagenesis ENU, EMS
• Phenotyping
• Collaborators
• Duke Univ. (lung function)
• Univ. of Pennsylvania (sleep)
• Boston Univ. (hypertension)

• Bioinformatics
• MutaJax
• Phenotype-specific
• genome maps
• Web site http//pga.jax.org
• Training Education
• Courses, VI Program
• Animal Distribution
• Archiving
• Administration

• Interactive with
• TREX PGA (gene arrays)
• PhysGen PGA (rats)

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Strain CharacterizationBeverly Paigen
Comprehensive phenotyping of 40 inbred strains
and 2 RI strains - SCP guidelines 10 each sex
per strain 10 weeks old - Data submitted to
Mouse Phenome Database (MPD) F2 Intercrosses
between strains differing in multiple phenotypes
- QTL analysis - Identification of
interacting loci (Churchill) Generation of
phenotype-specific genome maps - Graphic
representation of all QTL locations, mutants,
and candidate genes relevant to a particular
phenotype (atherosclerosis, hypertension,
obesity, etc)
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Phenotype-Specific Genome Map Hypertension
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N-ethyl-N-nitrosourea
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ES Cell Mutagenesis using EMS
Advantages over ENU 1. Eliminates injection of
G0, reducing the of generations from 3 to
2 2. Twice as many mutations can be
identified because germ cells from chimeras
will all be mutagenized.
(ICR191)
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G3 Phenotyping Protocol (Goal 4000 G3s/yr)
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1,300 mice/yr (wt)
2,700 mice/yr Begin Ath Diet (wt)
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Begin Atherogenic Diet (wt)
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G3 Protocol, continued
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2
4
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End Ath Diet At 15wks
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146
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What is a Mutant?
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What is Normal?
We use a 3-step approach to identifying
mutants - Non-mutagenized C57BL6/J (4 seasonal
groups) - G3 Averages (seasonal averages) -
Experts with experience EXAMPLE Glucose
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PhenotypingNewer Technologies

• Adaptation of clinical tests to mice
• Recently developed systems designed for testing
  mice non-invasively
• High-throughput (85 mice/week)

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Blood Coagulation Analyzer
Sample volumes cut in half, to 25µl of
plasma New protocols for mice New assays
not previously done for mice (ATIII)
Dade Behring, Marburg, Germany
Peters, et al. (2002) Physiol Genomics 11 185-193
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HLB18 Leukocytosis

• WBC 20.5 x 103 cells/ml (3.0-15.6) (m)
• Heritable
• Mapped x C3H (F2) to Chr. 12
• Crossing to C3H produced an anemia
• Currently available for distribution

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CCMSComprehensive Cage Monitoring System

• 3-day test
• VO2, VCO2, RER
• Food, water intake
• Rearing ambulatory activity

Columbus Instruments
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Assessing Sleep in MiceAllan Pack, U.
PennsylvaniaSubcontract PI, JAX PGA
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Sleep Parameters Ray Galante, UPenn
2 photoperiods Light and Dark For each
period we define total sleep (minutes) total
wake (min) sleep bouts average sleep bout
duration (min) average wake bout duration
(min) Overall, we calculate light/dark ratios
for sleep and wake
Light
Dark
L/D
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Sleep Phenotypes
SLEEP WAKE FRAGMENTATION CONSOLIDATION
(interrupted sleep) (uninterrupted sleep)
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Obstructive Sleep Apnea
Phenotype FO (more sleep wake bouts with
shorter duration, L D)
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OSA II Obese Models
Phenotype ESD ESO (less activity in light
and dark)
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Whole-body Plethysmograph
Measuring Lung Function Frequency, Tidal
Volume, Penh Baseline (saline) CO2
challenge Hypoxia challenge Methacholine
(5, 10, 20 mg/ml)
Buxco Electronics
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Empiric Derivation of Penh
Te Expiratory time (sec) Tr Relaxation time
(sec) PIF Peak inspiratory flow (ml/sec) PEF
Peak expiratory flow (ml/sec)
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Airways Hyperactivity
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Penh Pros and Cons

• Disadvantages
• Not derived from 1st physical prin
• Only correlates responsiveness
• Varies with breath timing, gas temperature, and
  humidity
• Unable to distinguish upper from lower airway
  obstruction

• Advantages
• Mice are conscious and viable
• Aerosolize bronchoconstrictor
• High throughput
• Reproducible

Julia Walker
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Airway Pressure Time Index (APTI)

• Relatively simple surgical preparation
• Fixed volume ventilator
• Resting tracheal pressure is measured
• Bronchoconstrictors cause increased tracheal
  pressure

Julia Walker
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APTI Surgical Preparation

• Anesthetize
• Cannulate trachea and ventilate
• Measure tracheal pressure
• Cannulate right external jugular vein
• Paralyze
• i.v. methacholine or vagus nerve stimulation

Julia Walker
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Airway Pressure Following Mch injection 150
mg/kg
APTI is the area under the peak pressure vs. time
curve
Julia Walker
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APTI

• Advantages
• relatively simple surgical preparation
• tracheal pressure in small species parallels
  changes in lung resistance
• good indicator of airway responsiveness as
  assessed by resistance measures
• Disadvantages
• cannot determine if changes are due to airway
  resistance or lung compliance
• terminal experiment

So far, 4/5 deviants tested show
airway hyperresponsiveness
Julia Walker
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Blood Pressure tail-cuff

• Visitech BP-2000
• Systolic BP, pulse
• Training days followed by measurement days (20-30
  meas/day)
• Salt-induced hypertension

Hypertension Mutants SBP elevated by 20-30 mmHg
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Blood Pressure Data
Randy von Smith
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Cardiac Ultrasound

• Sonos 5500 (Philips)
• 2-D, M-mode, Doppler
• Chamber dimensions
• Wall thicknesses
• Hemodynamics
• Transducers
• IOP/Linear (15-6 MHz band width)
• Sector (fusion band 5-12MHz)

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Conscious EKG

• HR (bpm)
• HR variability (bpm)
• P-R interval (ms)
• QRS complex (ms)
• QT segment

Mouse Specifics, Inc.
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Cardiac Function
2-D short axis view of left ventricle
Diastole
Systole
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Phenotypic Deviant DCM
Diastole
Systole
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Mutant Inbred Strain?

• Cardiac Ultrasound

• Anomalous LV pattern
• Short axis M-mode

• Anesthetic effect?
• Try conscious EKG

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(No Transcript)
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Check MPD!
Conclusion We have a true deviant!


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Obesity

• Weight
• 8, 12, 14 weeks of age
• Dual X-ray absorptiometry (DEXA)
• Normal weight at 12 weeks of age
• With increased body fat
• Increased weight at 12 weeks of age
• With increased body fat
• Overt phenotype present at wean
• Increased weight
• Increased body fat
• Leptin
• Diabetes Glucose, Insulin
• BMD, BMC

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Obesity Deviants

• Weight
• 8, 12, 14 weeks of age

• DEXA body fat
• 12 wks of age Ath diet 3 wks

Complementation shows Thor is a remutation of
Leprltdbgt
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Mutagenesis meets Phenotyping
HLBS Phenotypic Deviants April 2001 - July 15,
2003 30 Blood disorders (7) 21
Coagulopathies (3) 2 Thrombosis Risk 13
Blood Glucose (3) 87 Lipidopathies (9)
43 Body Fat (16) 23 Hypertensive (15)
25 Airway Hyperactivity (6) 11 Sleep (4)
8 Cardiac Dysfunction (3) 6 Bone (3)
evidence of heritability
High Throughput Phenotyping of G3 Mice Detects
Phenotypic Deviants
Total 269 HLBS deviants
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Testing for Heritability

Mutant m/m or m/
B6
X
We do NOT know the genotype!!!
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Mapping
Once a heritable mutant line is being
established, affected animals are available for
further analysis of the mutation that we suspect
causes the deviant phenotype. This includes
more invasive characterization of the
biology/physiology tissue harvesting for
gene microarrays (TREX PGA) thorough
histological work-up (Rod Bronson) genetic
mapping
Mapping Mate the mutant to a different inbred
strain that has about the same phenotype as B6.
Create 50-100 F2 animals and phenotype them all.
Perform a genome-wide DNA marker scan on the
highs and lows in the trait distribution to
find a single major locus that clearly influences
the phenotype. Knowledge of QTLs between B6 and
mapping strain will help!
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http//pga.jax.org
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Chemically Induced Mutants Page
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Phenotypic Deviant Report
Free Mice!


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Currently Available Models
Hypertension (8) Blood Leukocytosis
(1) Coming this summer! obesity, blood,
hypertension, cholesterol, lung/blood
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JAX PGA Participants

• Strain Characterization
• Beverly Paigen, Gary Churchill
• Mutagenesis
• Tim OBrien, Kim Meyers
• Phenotyping
• Karen Svenson
• Randy Smith
• Jeff Cash
• Phyllis Magnani
• Heather Ellis
• Beckie Durgin
• Laura Davis
• Holly Savage
• John Szwec
• Ann Plaud
• Linda Jorgenson
• Stacey Dannenberg
• Willson Roper
• Sarah Hyatt

• Bioinformatics
• Carol Bult
• Jie Sun
• Ranjan Jha
• Nazira Bektassova
• Kim Forthofer
• Archiving Distribution
• Larry Mobraaten
• Training Education
• John Macauley
• Administration
• Luanne Peters
• Martha Abbott

Collaborators Haralambos Gavras, Conrado
Johns, Boston U. Allan Pack, Ray Galante, U.
Penn David Schwartz, Julia Walker, Duke U.