Sponsored by: Participating Experts: Dr. Carl Lupica National Institutes of Health Baltimore, MD Dr. Kerry Ressler Emory University Atlanta, GA Dr. Rosalba Sacca Pfizer, Inc. Groton, CT Brought to you by the Science/AAAS Business Office 15 September, 2010 15 September, 2010 Beyond the Knockout Mouse Beyond the Knockout Mouse New Opportunities for Genetic Engineering in Animals New Opportunities for Genetic Engineering in Animals Webinar Series Webinar Series Science Science
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Sponsored by:
Participating Experts:
Dr. Carl LupicaNational Institutes of HealthBaltimore, MD
Dr. Kerry ResslerEmory UniversityAtlanta, GA
Dr. Rosalba SaccaPfizer, Inc.Groton, CT
Brought to you by the Science/AAAS Business Office
15 September, 201015 September, 2010
Beyond the Knockout MouseBeyond the Knockout MouseNew Opportunities for Genetic Engineering in AnimalsNew Opportunities for Genetic Engineering in Animals
Webinar SeriesWebinar SeriesScienceScience
"Beyond the Knockout Mouse: New "Beyond the Knockout Mouse: New Opportunities for Genetic Opportunities for Genetic
Engineering in Animals"Engineering in Animals"
Carl Lupica, Ph.D.Carl Lupica, Ph.D. NIH/NIDA IntramuralNIH/NIDA Intramural
Baltimore, Maryland, USABaltimore, Maryland, USA
Genetically engineered animals in Genetically engineered animals in neuroscience researchneuroscience research
1.1. Gene Deletion: The global Gene Deletion: The global ““knockoutknockout”” mousemouse
–– Useful for investigating the role of the missing protein targetUseful for investigating the role of the missing protein target–– Particularly important in behavioral pharmacology when good Particularly important in behavioral pharmacology when good
antagonists are not available.antagonists are not available.
–– Susceptible to problems of interpretation because of compensatioSusceptible to problems of interpretation because of compensation n for the deleted protein by physiological systems. for the deleted protein by physiological systems.
–– Redundant neural systems can also complicate interpretation.Redundant neural systems can also complicate interpretation.
–– Conditional knockouts can eliminate some of these problems Conditional knockouts can eliminate some of these problems because the timing of gene deletion can be controlled, thereby because the timing of gene deletion can be controlled, thereby limiting the amount of time for compensation to occur.limiting the amount of time for compensation to occur.
Genetic elimination (knockout) of the Genetic elimination (knockout) of the adenosine Aadenosine A
Genetically engineered animals in Genetically engineered animals in neuroscience research (contd.)neuroscience research (contd.)
•• PromoterPromoter--driven gene knockout, gene overexpression, or driven gene knockout, gene overexpression, or transgene expressiontransgene expression
–– Useful for targeting a knockout to a specific neuronal populatioUseful for targeting a knockout to a specific neuronal population.n.–– Useful for targeting gene overexpression to a specific neuronal Useful for targeting gene overexpression to a specific neuronal
population.population.–– Useful for labeling a specific neuronal population for selectionUseful for labeling a specific neuronal population for selection..
–– e.g. green fluorescent protein expression in druge.g. green fluorescent protein expression in drug--activated neuronal activated neuronal populations (driven by Cpopulations (driven by C--fos promoter).fos promoter).
–– e.g. deletion of e.g. deletion of Tfam Tfam gene in dopamine neurons (driven by gene in dopamine neurons (driven by dopamine transporter, DAT, promoter).dopamine transporter, DAT, promoter).
cc--fosfos promoter drives GFP expression promoter drives GFP expression in the transgenic mouse brainin the transgenic mouse brain
c‐fos promoter
FosGFPfusion protein
Neuronal Activation(cocaine)
GFP Alexa 568DIC Patch clamp
cc‐‐fosfos
promoter drives GFP expression in promoter drives GFP expression in the transgenic mouse brain: Advantagesthe transgenic mouse brain: Advantages
1.1. Identification of specific neurons activated by Identification of specific neurons activated by
abused drugs or other environmental stimuliabused drugs or other environmental stimuli
2.2. Identification of neuronal networks involved in Identification of neuronal networks involved in
3.3. Evaluation of physiological and biochemical Evaluation of physiological and biochemical
changes in highly relevant neuronal populationschanges in highly relevant neuronal populations
cc‐‐fosfos
promoter drives GFP expression in promoter drives GFP expression in the transgenic mouse brain: Limitationsthe transgenic mouse brain: Limitations
1.1. Limited temporal expression of Limited temporal expression of cc‐‐fos fos = limited window = limited window
for experiments (~ 6 hours)for experiments (~ 6 hours)
2.2. Activation of Activation of cc‐‐fos fos must immediately precede tissue must immediately precede tissue
preparation. Longer lasting signal neededpreparation. Longer lasting signal needed
3.3. Mouse models not as widely used as rat, not as Mouse models not as widely used as rat, not as
applicable as nonapplicable as non‐‐human primateshuman primates
Dopamine transporter promoter drives Dopamine transporter promoter drives deletion of mitochondrial transcription factor A deletion of mitochondrial transcription factor A
((Tfam)Tfam)
gene in micegene in mice
• All dopamine neurons lack Tfam• A “double transgenic” model• Mitochondrial gene replication is halted• Mitochondrial energy production fails• Results in a Parkinson’s disease-like behavioral
and neurochemical phenotype (“MitoPark mouse”)
• Closely models a human disease despite targeting a gene not thought to be involved in the disease
Mouse 3:Mouse 3: Cross of 1 and 2: Cross of 1 and 2: In DA neuronsIn DA neurons cre are made,cre are made,excise TFAM, and reconnect the DNA strand, leaving only 1 LoxP sexcise TFAM, and reconnect the DNA strand, leaving only 1 LoxP siteite
TFAM
TFAM
Mouse 1:Mouse 1: Gene of interest flanked by two similarly oriented LoxP sitesGene of interest flanked by two similarly oriented LoxP sites
CreCre‐‐loxP gene excision of loxP gene excision of TfamTfam
only in DA neuronsonly in DA neurons
Mouse 2:Mouse 2: Cre recombinase expressed under specific promoter:Cre recombinase expressed under specific promoter:
2.2. More costly in terms of money and timeMore costly in terms of money and time
3.3. Careful selection of control genotype requiredCareful selection of control genotype required
4.4. Mouse models not as widely used as rat, not as Mouse models not as widely used as rat, not as
applicable as nonapplicable as non‐‐human primateshuman primates5.5.
The time course of the disease phenotype is to some The time course of the disease phenotype is to some
degree dictated by the lifespan of the animaldegree dictated by the lifespan of the animal
•• Chronic neurodegenerative diseases could benefit from Chronic neurodegenerative diseases could benefit from
the use of transgenic model organisms with longer life the use of transgenic model organisms with longer life spans than rodents.spans than rodents.
Acknowledgements ••
National Institutes of Health/ National Institute on Drug AbuseNational Institutes of Health/ National Institute on Drug Abuse
Intramural Program Baltimore, MD, USAIntramural Program Baltimore, MD, USA
••
Dr. Barry Hoffer, NIDADr. Barry Hoffer, NIDA••
Dr. Bruce Hope, NIDADr. Bruce Hope, NIDA
••
Dr. Eisuke Koya, NIDADr. Eisuke Koya, NIDA••
Dr. Cristina BDr. Cristina Bääckman, NIDAckman, NIDA
••
Dr. Susan Masino, Trinity CollegeDr. Susan Masino, Trinity College
••
Dr. Lars Olson, Dr. NilsDr. Lars Olson, Dr. Nils‐‐GGööran Larsson, Dr. Dagmar Galter, and ran Larsson, Dr. Dagmar Galter, and colleagues Karolinska Institute, Stockholm, Sweden colleagues Karolinska Institute, Stockholm, Sweden
••
Kriss Knestaut, M.S. NIDAKriss Knestaut, M.S. NIDA‐‐IRP Transgenic Breeding DirectorIRP Transgenic Breeding Director
Sponsored by:
Participating Experts:
Dr. Carl LupicaNational Institutes of HealthBaltimore, MD
Dr. Kerry ResslerEmory UniversityAtlanta, GA
Dr. Rosalba SaccaPfizer, Inc.Groton, CT
Brought to you by the Science/AAAS Business Office
15 September, 201015 September, 2010
Beyond the Knockout MouseBeyond the Knockout MouseNew Opportunities for Genetic Engineering in AnimalsNew Opportunities for Genetic Engineering in Animals
Webinar SeriesWebinar SeriesScienceScience
Beyond the KO Mouse
Rosalba Sacca Science Webinar
September 15, 2010
Genetically Modified Models (GeMM) Research Center of Emphasis
Mouse models are an established critical tool in drug discovery
Validate biology of target of interest (unprecedented mechanisms).
Screening: Test compound pharmacology and selectivity, screening tool
Control variability: Use inbred strains to our advantage, certain strains perform better in certain assays and controlled environment
Statistical power: can do large studies, ethical concerns in use of larger animals, expense.
Genetic manipulation: make multiple genetic manipulations at and better understand pathways.
Limitations:
Time, Expense, Translation
Labor intensive phenotypic analysis
KO Mouse Models: Extracting Value
How can we extract the most value from KO mice?
Comprehensive Phenotyping
WHY?
About 50% of Pfizer KOs yield unexpected phenotypes or no phenotype
Systematic identification of new phenotypes and potential adverse effects will maximize our investment in KO mice
50 KO & 50 WT mice evaluated in ~60 assays carried out sequentially, covering ~12 Therapeutic Areas in 17 weeks
All data deposited in database available to all
Partnership with Xenogen Biosciences
Bioluminescence Imaging
Use of Bioluminescence Imaging to Obtain Increased Value from Mouse Models
Platform mouse line to evaluate CRE- activated gene expression resulting from CREB-mediated signaling events.
Cyclic-AMP Response Element (CRE) Luciferase Reporter Transgenic Mouse
CRECRE CRECRE CRE CRE E1b Luciferase pA
Inhibition of PDE Elicits Robust StriatalSignal In CRE Tg Mice
Baseline
16 hr.
Vehicle treated treated Vehicle treated treated
Liver Luminescence in Response to Fasting and GlucagonBaseline Post Treatment
1) GenesHuman genomic Knock In
2) Somatic Cells‐
Human Peripheral Blood
3) Stem cells/Organs‐
Human Cord Blood‐
Human Liver‐
Pancreatic Endoderm
Humanized Mice
Frese and Tuveson Nat .Rev.Cancer. 2007
Generation of Humanized TPOr Model
Thrombopoietin (TPO) receptor is primary factor in growth and differentiation of mature plateletsLead compound is species specific : No available animal modelsNeed for a humanized mouse model
Human Receptor
Mouse Receptor
Humanized mouse
Humanized TPOr Model
1153 1240 37272819 42961390
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Day 0 Day 4 Day 5 Day 0 Day 4 Day 5
Pla
tele
ts K
/µ
L
Wild Type Knock In
compound 60 mg/kg
32%
49%
Essential tool for testing lead compounds
Tool to Establish Exposure vs. Therapeutic Effect
0
10
20
30
40
50
60
0 15 30 60
[PF-cmpd mg/kg] s.c. days 0,1
% p
late
let
incr
ease
10%
16%
40%
52%
T i m e ( h )
0 1 0 2 0 3 0 4 0
PF-0
2542
376
plas
ma
leve
ls (u
g/m
l)
0 . 0
0 . 1
0 . 2
0 . 3
0 . 4
0 . 5
0 . 6
•in vivo tool for establishing human dose projections
•Potential use for toxicology evaluation
Humanized Mouse Models: Transplantation of Human Cells
Human Adult Peripheral Blood transplantation
Human Cord Blood transplantation
Human liver hepatocytes transplantation
NSG Host
300ml
Blood
Ficoll
2.106
HPBMCGraft versus Host Disease
Platform
Cord
Blood
3.104
CD34+ cells
4-6 weeks
NeonateNSG Host
Mature T cell onlyGVHD onset rate / h.MAbs
2-6 monthsLong-term,
Human Immune System platform
Immature lymphoid cells
100 rad
Humanized Liver Model (Yecuris)Normal human hepatocytes take residency in mouse liver
Pancreatic Progenitors Mature to Functional Islets In Vivo
Human Islet Graft Implanted 360 days
hESC-PE GraftImplanted 377 days
Grafts show hallmarks of bona fide human islets
GlucagonSomatostatinInsulin
Figure from
28
What’s next? Beyond the Mouse…
ZebrafishAdvantages:• Small size (2-3 embryos / 384 well
plate)• High fecundity (~300 eggs / mating)• Transparent: easily visualize
development • Low maintenance costs ($0.01/day)• Major organ systems similar to
mammal• Easily amenable to genetic
manipulation• Compatible with bioimaging platform
Disadvantages:• Translation & Bioavailability
Example: Muscle Toxicity:
Atrogin-1 – Luc2 reporter construct for drug induced myopathy
NEED: Fill the gap between in vitro assay & rodent histopathology assay
IMPACT: ‘Lower $$ and HT of in- vitro approach in more relevant in- vivo model’
Pfizer Aquarium
Genetically Modified Rat
Zinc Finger Nuclease (ZFN) – Contains 2 domains:
Nuclease domain of FokIDesigner zinc finger protein
– Cleaves as a dimer– May be engineered to cleave
virtually any sequence– Effectively a “designer
restriction enzyme”
RAT KO
Preferred preclinical model for R&D
Until recently, inability to manipulate rat genome due to lack of germline competent ES cells
Progress to date :
Sequencing of rat genome
ZFN technology
Germline competent rat ES cell lines (Tong et al. 2010)
Dr. Carl LupicaNational Institutes of HealthBaltimore, MD
Dr. Kerry ResslerEmory UniversityAtlanta, GA
Dr. Rosalba SaccaPfizer, Inc.Groton, CT
Brought to you by the Science/AAAS Business Office
15 September, 201015 September, 2010
Beyond the Knockout MouseBeyond the Knockout MouseNew Opportunities for Genetic Engineering in AnimalsNew Opportunities for Genetic Engineering in Animals
Webinar SeriesWebinar SeriesScienceScience
"Beyond the Knockout Mouse: New "Beyond the Knockout Mouse: New Opportunities for Genetic Opportunities for Genetic
Engineering in Animals"Engineering in Animals"
Kerry J. Ressler, M.D., Ph.D.Kerry J. Ressler, M.D., Ph.D. Howard Hughes Medical InstituteHoward Hughes Medical Institute
Emory UniversityEmory University Atlanta, GAAtlanta, GA
Genetic Models to Study BehaviorGenetic Models to Study Behavior1. The importance of inducible knockout systems in studying behavior
2. Region-specific Cre transgenic driver lines and lentivirus-driven Cre to delete Brain Derived Neurotrophic Factor (BDNF) in cortex and hippocampus
3. Amygdala specific knockout of Beta-catenin and memory consolidation
4. Cell-type specific promoters to knockout genes in specific neurons / circuits
5. Use of targeted knockin reporter lines to study sensory system plasticity
6. Humanized mouse knockin to study a characterized BDNF polymorphism
7. Knockout, followed by cortex-specific replacement of 5HT1a and behavior
8. Other new transgenic and targeted knockin models (e.g. primate and vole)
9. Limitations of current genetic systems
Use of Inducible Genetic Systems in the Use of Inducible Genetic Systems in the Study of Learning, Memory, and BehaviorStudy of Learning, Memory, and Behavior
• Learning and memory processes utilize highly dynamic and specific brain circuits
• A primary research goal is to understand the molecular / cellular / circuit mechanisms which underlie emotional memory (psychiatric disorders) and declarative memory (degenerative disorders / dementia)
• Transgenics and standard knockouts are often inadequate because:• would ideally make spatially / regionally restricted gene deletions• need temporal-specific deletions, e.g. only in adult or only before or after learning event.
• Ideal models rely on inducibility of a gene or gene pathway or inducible gene knockouts within a restricted brain region
Some Limitations of Current SystemsSome Limitations of Current Systems (from behavioral neuroscience perspective)
• Limitations of mice in human disease, particularly complex disease and behavior
• In general, important to have more easy access to a variety of species that can be optimally examined related to their behavior / phenotype / tissue type etc.
• Importance of inducibility and remaining relatively long lag-time with inducible deletion or expression
• Need better tools to control cell-type and regional specificity of expression in CNS
• Improvements needed in combining human genetic variant data with mouse genetic tools
• Need better MRI or PET sensitive genetic ligands to combine functional imaging with behavior and genetics
NSF, NSF, BurroughBurrough’’ss Wellcome Fund, NARSAD, ADAAWellcome Fund, NARSAD, ADAA•• Elizabeth BinderElizabeth Binder•• BekhBekh BradleyBradley•• Tanja JovanovicTanja Jovanovic•• Joe CubellsJoe Cubells•• Kristie MercerKristie Mercer•• Alicia SmithAlicia Smith•• Kimberly Kimberly KerleyKerley•• Michael DavisMichael Davis
–– David WalkerDavid Walker–– KarynKaryn MyersMyers–– KwokKwok--Tung LuTung Lu
•• Donald RainnieDonald Rainnie•• Barbara Barbara RothbaumRothbaum•• Michael OwensMichael Owens
•• Dennis C. ChoiDennis C. Choi•• Scott HeldtScott Heldt•• Kim Maguschak Kim Maguschak •• Seth JonesSeth Jones•• Elizabeth MartinElizabeth Martin•• JasmeerJasmeer ChhatwalChhatwal•• Lisa Lisa StanekStanek--RattinerRattiner•• Amy MahanAmy Mahan•• Georgette Georgette GaffordGafford•• Aaron JasnowAaron Jasnow•• LipingLiping MouMou
Sponsored by:
Participating Experts:
Dr. Carl LupicaNational Institutes of HealthBaltimore, MD
Dr. Kerry ResslerEmory UniversityAtlanta, GA
Dr. Rosalba SaccaPfizer, Inc.Groton, CT
Brought to you by the Science/AAAS Business Office
15 September, 201015 September, 2010
Beyond the Knockout MouseBeyond the Knockout MouseNew Opportunities for Genetic Engineering in AnimalsNew Opportunities for Genetic Engineering in Animals
Webinar SeriesWebinar SeriesScienceScience
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Brought to you by the Science/AAAS Business Office
15 September, 201015 September, 2010
Beyond the Knockout MouseBeyond the Knockout MouseNew Opportunities for Genetic Engineering in AnimalsNew Opportunities for Genetic Engineering in Animals