2

TCP mouse model production and phenotyping for functional annotation of the

genome

2LS

1. Genome Sequence Human & mouse ~95% homology

International Knockout Mouse Consortium

(IKMC) 2. Gene Mutations in mES cells

20,000 protein-coding genes

NorCOMM EUCOMM KOMP1 TIGM

CREATE Consortium

3. Tools for Translation Research Cre driver lines for conditional expression

(2011-2015)

NorCOMM2LS EUCOMMTOOLS

International Mouse Phenotyping Consortium

(IMPC)

4. Identification of Disease Models Phenotyping 20,000 mutant mouse lines

(2011-2020)

NorCOMM2 DTCC KOMP2

2

1. Genome Sequence Human & mouse ~95% homology

International Knockout Mouse Consortium

(IKMC) 2. Gene Mutations in mES cells

20,000 protein-coding genes

NorCOMM EUCOMM KOMP1 TIGM

CREATE Consortium

3. Tools for Translation Research Cre driver lines for conditional expression

(2011-2015)

NorCOMM2LS EUCOMMTOOLS

International Mouse Phenotyping Consortium

(IMPC)

4. Identification of Disease Models Phenotyping 20,000 mutant mouse lines

(2011-2020)

NorCOMM2 DTCC KOMP2

2

1. Genome Sequence Human & mouse ~95% homology

International Knockout Mouse Consortium

(IKMC) 2. Gene Mutations in mES cells

20,000 protein-coding genes

NorCOMM EUCOMM KOMP1 TIGM

CREATE Consortium

3. Tools for Translation Research Cre driver lines for conditional expression

(2011-2015)

NorCOMM2LS EUCOMMTOOLS

International Mouse Phenotyping

Consortium (IMPC)

4. Identification of Disease Models Phenotyping 20,000 mutant mouse lines

(2011-2020)

NorCOMM2 DTCC KOMP2

2

Toronto Centre for Phenogenomics

Two Infrastructure Support Programs • TCP Research & Facility Operations

– Animal holding and facilities for PI-driven research– Animal holding to support Research Programs at TCP

• TCP Transgenic Core– Transgenic & knockout production, embryo biology

Three Research Programs • Centre for Modeling Human Disease (CMHD)

– Random & targeted mutagenesis– Physiology and Neurobiology phenotyping– Pathology (Gross, Histology, Molecular) phenotyping

• Canadian Mouse Mutant Repository (CMMR)– Cryopreservation & archiving services for mouselines– IVF for recovery and storage of mouselines– National repository for IKMC

• Mouse Imaging Centre (MICe)– Multi-modality mouse imaging collaborations

NorCOMM2 Project Objectives 2

NorCOMM2 Production & Cryopreservation

Production Year

TCP Morula Aggregation

MRC Harwell Blast Injection

Total

Y01 65 66 111

Y02 85 82 132

Y03 0 82 132

Y04 0 99 99

Y05 0 0 0

150 330 480

NorCOMM2 Phenotyping

Phenotyping Year

TCP Adult HOM / Adult HET / Subviable

MRC Harwell Adult HOM

Total Adult HOM +

Subviable

Y01 0 0 50

Y02 45 / 30 / 30 80 130

Y03 45 / 30 / 30 82 132

Y04 0 82 82

Y05 0 86 86

150 330 480

Harwell PM Centre Lead: Kent Lloyd

Gene Nomination Mouse Lines Gene Nomination

TCP PMs Ann Flenniken Lauryl Nutter

Mouse Lines

• Gail Martin, University of San Francisco• Expertise in developmental biology and using the mouse as a model for human

disease and development

• Phil Soriano, Mount Sinai School of Medicine• Expertise in developmental biology, neurobiology, stem cell and cancer biology, as Resource Requests Phenotype Data

Canadian Researchers and BioPharma MRC Harwell

Resource Requests Phenotype Data as mouse mutagenesis and phenotyping

Scientific Advisory Board (SAB)

SMEC Leads: McKerlie, Brown

Adamson, Bubela, Rossant, Weaver, PMs

IMPC

NIH DTCC Production Centre

Lead: Kent Lloyd

NIH DTCC Phenotyping

Other IMPC Projects

Scientific Advisory Board (SAB)

• Phil Avner, CNRS Institut Pasteur• Expertise in the generation, characterization, and use of mouse models for the study of

development and disease

• Neal Copeland, A*Star Institute of Molecular and Cell Biology• Expertise in cancer genetics and the use of mouse models to study various disease

processes

• Dan Drucker, Samuel Lunenfeld Research Institute• Expertise in mouse models of diabetes, obesity, and intestinal disorders, in design and

testing of new drugs and therapeutics

well

NorCOMM2 Project Coordination 2

NorCOMM2 Project Workflow 2

IKMC ES Cell Validation Expansion , Confirmation ,

Pathogen Testing

Toronto Harwell

Breeding & Expansion to Produce Phenotyping Cohorts

Phenotyping Pipeline Homozygous lethality screen

Fertility sc reen Cryopreservation

Toronto Harwell

Heterozygote breeding of homozygous

lethal lines

E 9 or E 14 embryo imaging

Toronto

Blastocyst Microinjection

Toronto

Chimeras - > F 1 HETs Up to 3 clones per line

Sperm , embryos , live mice to CMMR or EMMA

repository Clones for

Conversion

Harwell

Morula Aggregation

NorCOMM2 Gene Selection 2

2% (12)

0

Genes 427 206 160 96 30

Nominated TCP list Knock-out f irst alleles

Assigned to TCP

In ES cell QC

100

200

300

400

500 206

No IKMC ES cells

Not in MGI

IKMC ES cells

73% (427)

47

96

10 10

86

(142)

020

46 4024%

60

80100

120140

132

Genes Nominated to NorCOMM2.org 2

• 581 unique gene nominations from Canadian scientists and 3 consortia• Ontario Institute for Cancer Research• Cystic Fibrosis Modifier Gene Consortium• Lysosomal Storage Research Group

NorCOMM2 Gene Selection 2

NorCOMM2 Production Flowchart 2

Validated ES Cell

Aggregation

>50% ♂ chimera

♂ ♀

Next clone NO of same gene

(3 max)

YES

chimera × B6N

GLT

YES

Cre Excision

Establish Genetm1b mouse line (deletion)

Generate cohort for phenotyping

NO

♂ Genetm1

(knockout first)

Sperm Cryo

a

YES

NorCOMM2 Adult Phenotyping 2

♂ ♀

Genotype at P14 (expect 25% Homozygotes)

HET × HET

>10% Homozygotes <10% Homozygotes

Neonatal Lethal Pipeline

Embryonic Lethal Pipeline Viable HOM Subviable HET Subviable

9

Mod

ified

SH

IRPA

Ope

n Fi

eld

Gri

p St

reng

th

Gai

t A

naly

sis

Aw

ake

ECG

Intr

aper

iton

eal G

luco

se T

oler

ance

Tes

t

Aud

itor

y Br

ain

Stem

Res

pons

e

DEX

A (B

one

Den

sito

met

ry)

X-ra

y

Slit

Lam

p

Oph

thal

mos

cope

Hem

atol

ogy

Clin

ical

Flo

w C

ytom

etry

Clin

ical

Blo

od C

hem

istr

y

Insu

lin B

lood

Lev

el

Imm

unog

lobu

lin C

once

ntra

tion

Gro

ss P

atho

logy

His

topa

thol

ogy

DN

A a

nd T

issu

e Ba

nkin

g

Hea

rt W

eigh

t

Calo

rim

etry

12 10 14 16 15

Terminal

Bleed

Weight Curve – 4 wk to 16 wk 7 M + 7 F Adult Mice from Viable or Subviable Lines

Dys

mor

phol

ogy

Aco

usti

c St

artl

e /

PPI

11

Hot

Pla

te o

r vo

n Fr

ey F

ilam

ent

Test

Uri

naly

sis

13

Dys

mor

phol

ogy

16

N=7

M N

=2M

+ 2

F

N=2

M +

2F

N=2

M +

2F

N=5

M +

5F

NorCOMM2 Neonate & Embryo Phenotyping 2

8 Neonates

P1-P7

Histopathology

♂ HET × ♀ HET

Neonatal Lethal

Genotype at P0-P1 (expect 25% Homozygotes)

>10% Homozygotes <10% Homozygotes

Embryonic Lethal

♂ HET × ♀ HET

Dissect and genotype at E14.5 (expect 25% Homozygotes)

>10% Homozygotes <10% Homozygotes

♂ HET × ♀ HET

Dissect and genotype at E9.5 (expect 25% Homozygotes)

>10% Homozygotes <10% Homozygotes

Lethal <E9.5

Dis

sect

ion

& g

ross

mor

phol

ogy

(em

bryo

s &

pla

cent

ae)

His

topa

thol

ogy

(em

bryo

s &

pla

cent

ae)

Embr

yo μ

CT

(iodi

ne s

tain

ing)

E14.5

8 Embryos

Embr

yo O

PT

His

topa

thol

ogy

(em

bryo

s &

pla

cent

ae)

Dis

sect

ion

& g

ross

mor

phol

ogy

(em

bryo

s &

pla

cent

ae)

E9.5

8 Embryos

Da t abase

Germplasm

Wet tissue Histology

Phenotype

Archive

Archive

Frozen tissue

Pathology Phenotyping at TCP 2

Da t abase

Embedded tissue

Fixed tissue

Phenotype

Archive

Archive

Tissue

Report

Pathology Phenotyping at TCP 2

Histopathology Screen Pilot Project

Project Design 1. Sanger MGP tissue collection

• 30 tissues collected from each of 2M + 2F, fixed in 10% NBF, then embedded in 14 tissue blocks

2. TCP tissue preparation • Cut 5μm sections from each block and stain with H&E

3. TCP tissue evaluation • Complete histopathological evaluation with MAID and MPath ontology and definitive morphological

diagnosis(es) for 2M + 2F per mouse line (10M + 10F for WT controls) • Image-enabled histopathology summary for each mouse line

4. Mouse line selection • 30 mouse lines chosen randomly from Sanger MGP pipeline with no in-life phenotype • 20 mouse lines chosen randomly from Sanger MGP pipeline with one or more in-life phenotypes

2

Histopathology Screen Pilot Project 2

Preliminary Results

Mutant lines with in-life phenotype Mutant lines with no in-life phenotype

Cohort Number of mouse lines

Spontaneous / Incidental findings(s)

Significant finding(s) associated with in-

life phenotype

Significant finding(s) unlikely associated

with in-life phenotype

Significant finding(s) likely association

with genotype

Significant finding(s) unlikely association

with genotype

Significant finding(s) with

unannotated gene

WT 2 2 - - - - -

In-life Phenotype 15 15 11 4 - - -

No in-life Phenotype 12 8 - - 4 0 0

29

So far: 1. Good concordance in % of mice with Spontaneous / Incidental Findings between WT and Mutants

• Most are B6-related (hydrocephalus, microphthalmia) or high fat diet-related (hepatic lipidosis,gastritis)

2. In 11 / 15 lines (73%) with in-life phenotype we are contributing tissue-level phenotype data

3. In 4 / 12 lines (33%) with no in-life phenotype we have identified significant findings likely associated withgenotype based on gene annotation

2 Histopathology Screen Pilot Project

Preliminary Results

Go Annotation (Biological Processes) Activation of MAPK pathway

Angiogenesis

Morphological Diagnosis(es) Hibernoma (2 of 4)

WT brown fat (x400) Mutant brown fat (x400)

2 Histopathology Screen Pilot Project

Preliminary Results

Go Annotation (Biological Processes) Cell differentiation Fatty acid metabolism Lipid metabolism Negative regulation of sequestering Triglyceride

Morphological Diagnosis(es) Diffuse sertoli cell vacuolation (2 of 2 males)

WT seminiferous tubules (x400) Mutant seminiferous tubules (x400)

2

Acknowledgements

• CMHD Phenotyping– Lee Adamson– Jane Aubin– Ann Flenniken– Lucy Osborne– Janet Rossant– Dwayne Barber

• CMHD Pathology Core– Hibret Adissu– Pat Feugas– Lily Morikawa– Susan Newbigging– Yanchun Wang– Qiang Xu– Napoleon Law– Yingchun Zhu

• CMMR– Lauryl Nutter– Joanne Roberti– Qing Fan

• RP-LIMS– Nora Jones– Faustino Fleitas– Susan Justin– Dimple Dhawan

• TCP Tg Core & Facility– Betty-Jo Edgell– Lise Phaneuf– Marina Gertsenstei

• Mouse Imaging Centre– Mark Henkelman– John Sled– Jason Learch

• NorCOMM2LS– Tania Bubela– Geoff Hicks– Andras Nagy– Janet Rossant

n

• Funding Agencies– Genome Canada– Ontario Genomics Institute– Ontario MRI– CIHR– NIH– Canada Foundation for Innovation

• MRC Harwell– Steve Brown– Tom Weaver

2LS