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.

–– Background strain interactions: e.g. cannabinoid CB1 receptor Background strain interactions: e.g. cannabinoid CB1 receptor knockout mice C57BL6 mouse strain demonstrates diminished knockout mice C57BL6 mouse strain demonstrates diminished learning, CD1 learning, CD1 ““SwissSwiss”” mouse strain demonstrates enhanced mouse strain demonstrates enhanced learning.learning.

–– 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

11

receptor permits receptor permits  cannabinoid signalingcannabinoid signaling

0.25 mV5 ms

0 10 20 30 40 500

50

100

Adenosine A1+/+

Adenosine A1-/-

WIN55,212-2 (500 nM)

Time (min)

fEPS

P Sl

ope

(% B

asel

ine)

A1+/+ A1-/-

Control

WIN WIN

A.

B.

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 

mediating drugmediating drug‐‐induced behaviorsinduced behaviors

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:

CreDAT promoter

DdTtDdTt DDttDDtt

XXDdttDdtt

““Mitopark”Mitopark”(2)(2)

DdTtDdTt

DDttDDttDDTtDDTt ““Controls”Controls”

DDTTDDTT

MitoPark mouse breeding SchemeMitoPark mouse breeding Scheme

DdTTDdTT DDttDDtt DdTtDdTt

XX

D= normal DATD= normal DATd = DATd = DAT‐‐crecre

T= normal TfamT= normal Tfamt = Tfamt = Tfam‐‐loxPloxP

(1)(1)

DAT promoter driven deletion of  mitochondrial Tfam

gene: Advantages: Advantages

1.1. Relatively Relatively ““normalnormal””

development of a human development of a human 

neurodegenerative disease neurodegenerative disease 

2.2. Does not require the use of neurotoxins, as in Does not require the use of neurotoxins, as in 

other Parkinsonother Parkinson’’s disease modelss disease models

3.3. The slower time course of the modeled disease The slower time course of the modeled disease 

permits evaluation of potentially beneficial permits evaluation of potentially beneficial  therapeutic interventionstherapeutic interventions

DAT promoter driven deletion of  mitochondrial Tfam

gene: Limitations: Limitations

1.1. Complex breeding schemeComplex breeding scheme

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

–Mission / Key Objectives• Develop & deliver genetically modified

animal systems for drug discovery (KI, KO, TG)

• Generate & deliver cell types differentiated from stem cells for target discovery, assay development and compound screening

• Use bioluminescence imaging capabilities to deliver improved target choice

• Humanized Mouse Transplantation Models

Neurons

GFAP+Astrocytes MAP2ab+Neurons

Neurons

GFAP+Astrocytes MAP2ab+Neurons

Genetically Modified Mouse Models

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)

Acknowledgements

Melanie AllenAmy Baumann

Bill BlakeChris Donahue

Regis DoyonnasSandra EngleAmy Friedrich

Shawn HallowellKeith Haskell

Ananya IndurtiKen Miller

Diane NadeauThao NguyenWenning Qin

Rosalba SaccaJeff Stock

Mark ThiedeMathew Wen

Thad Wolfram

TPO studiesPaul Lira

Robin NelsonSandra McCurdy

Sharon Ripp

GeMM RCoE

WWCM Cedo BagiCathy AndersenRich PeroRao VaradaJacinda Sortwell

DSRDJiri AubrechtBill ReaganDeborah KitchelSharon SokolowskiLeslie ObertDean WilkieTom BrownCathy TaborJohn KreegerMeg Wilhelms

Regenerative Medicine John McNeishMike Rukstalis

Neuroscience Research UnitRobin KleimanSusan Bove

Diabetes Research UnitChris Sallato

Robert DulleaGus Gustafson

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"

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

Amygdala

ILPFC

PLPFC

Hipp.

External sensoryExternal sensoryInternal memoryInternal memory

StressStressfearfear

Resilience / Resilience / AversionAversiontolerancetolerance

TrkB

bdnf

bdnf bdnf

Role of BDNF Role of BDNF ––

TrkB interactions in TrkB interactions in Cortical Cortical ––

Hippocampal Amygdala Hippocampal Amygdala 

circuit mediating emotioncircuit mediating emotion

Cre Recombinase

Choi et al., PNAS, 2010

Maguschak et al., Nature Neurosci, 2008

Martin et al., Molecular Psychiatry, 2010

Jones et al., JNeurosci, 2008

Soliman

et al., Science

2010

Weisstaub et al., Science, 2006

Yang et al., Nature, 2008

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

Acknowledgements:Acknowledgements: HHMI, HHMI, NIMH/NIH (MH069884, MH071537)NIMH/NIH (MH069884, MH071537)

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

Look out for more webinars in the series at:

www.sciencemag.org/webinar

For related information on this webinar topic, go to:

www.sageresearchmodels.com

To provide feedback on this webinar, please e‐mail

your comments to webinar@aaas.org

Sponsored by:

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