The automated mouse: Systematic generation of disease models

Nadia Rosenthal

EMBL Mouse BiologyMonterotondo, Rome

Harefield Heart Science CentreImperial College London

Australian Regenerative Medicine InstituteMonash University, Melbourne

The automated mouse:Systematic generation of disease models

European Conditional

Mouse Mutagenesis

Program

EUCOMM

Developing mouse mutants for most of the genes in the

mouse genome in ES cells

EUMORPHIA

Development and standardisation of mouse phenotyping

platforms

EUMODIC - European Mouse Disease Clinic

Undertake a major pilot programme to utilise mouse mutant

production and standardised phenotyping platforms for the

analysis of a large number of mouse mutants

European mouse mutagenesis and

phenotyping programs


Mouse Mutagenesis

ProgramGene knockout vs. conditional mutatagenesis

geneX del

Cre

geneX cond

Promoter Yy Cre

Gene X is inactivatedby Cre ONLY in tissue Y

X

geneX del

Gene X is inactivatedin all tissues

geneX

Targeted deletionof critical exon

Cre-mediated excisionof critical exon

Knockout Conditional knockout


Mouse Mutagenesis

Program

» Generation of up to 12,000 conditional gene trap mutations in EScells

» Generation of up to 8,000 targeted conditional mutation

» Establishment of up to 320 mutant mouse lines

» Archiving and distribution of vectors, ES cells and mutant mice

» Integration of EUCOMM effort with KOMP(US) and NorCOMM(Canada)

EUCOMM Objectives:


Mouse Mutagenesis

Program

EUCOMM – KOMP – NorCOMM interactions:

1. Complementation of resource generation (gene targeting, gene

trapping)

2. Common presentation of resources to the scientific community

– integration into IMSR, Ensembl, and NCBI databases

3. Common distribution centers for EUCOMM-KOMP-NorCOMM

material


Mouse Mutagenesis

Program


Mouse Mutagenesis

Program

University of DresdenF. Stewart

National Research CouncilG. Tocchini-Valentini

Institute Clinique de la Souris (ICS)J.Auwerx, P. Chambon

EMBLN. Rosenthal

Medical Research CouncilS. Brown

Deutsches Ressourcenzentrumfür Genomforschung (RZPD)A. Hörlein

GSF National Research CenterW. Wurst (coordinator), M. Hrabé de Angelis

Wellcome Trust Sanger InstituteA. Bradley (coordinator), W. Skarnes, P. Liu

University of FrankfurtH. von Melchner

Max-Planck-Institute of Molecular GeneticsP. Ruiz


Mouse Mutagenesis

Program

Subprojects (SP) and Work Packages (WP)

Structure of EUCOMM


Mouse Mutagenesis

Program

13,000 genes

mutant mice

LacZ-taggedconditional mutant ES

cell resource

A dual resource of targeted ES cellsand modular targeting constructs

Modular targetingconstructs &

cassettes

additional alleles

8,000 genes (EUCOMM)5,000 genes (KOMP)

EUCOMMEUCOMMKOMPKOMP


Mouse Mutagenesis

ProgramGene target vs. trap

geneX

geneX del


geneX


Insertion of a new codingregion, disrupting gene

Target Trap

Reporter on insert“traps” readout ofgene expressionpattern


Mouse Mutagenesis

Program


Mouse Mutagenesis

Program

Conditional gene trap vectors

rFlipRosageo

frt

loxP

F3

lox5171

SA geo pALTR LTR

frt F3

loxP lox5171

rFlipRosaCeo

frt

loxP

F3

lox5171

SALTR LTR

frt F3

loxP lox5171

pACeo

TMSA

Schnütgen et al, PNAS, 102, 7221, 2005


Mouse Mutagenesis

Program

SA geo pAEx 1 Ex 2

SAgeopA

Ex 1 Ex 2

SA geo pAEx 1 Ex 2

mutation

no mutation

mutation

+ Cre

+ FLPe


Mouse Mutagenesis

Program

Validation of conditional gene trap vector in vitro

t

inv

re-inv

wt t

wt t inv re-inv

pol II

SA geo pAE1 E3 E5

inv re-inv

endo fus endo fus endo fus endo fusRBBP7

wt t inv re-inv

lamin A

+ FLPe + Cre

+ FLPe + Cre


Mouse Mutagenesis

Program

Present status of gene trap:

» Production of 44,800

» Generation of 22,000 clones

» Sequence of 2,000 – 1,212 clones successfulprocessed – representing approximately 700 differentgenes

» Success rate of sequencing and annotation ofproducts is approximately 63%

» Present weekly production rate: 4,000 clones perweek


Mouse Mutagenesis

ProgramGene target vs. trap

geneX

geneX del


geneX


Insertion of a new codingregion, disrupting gene

Target Trap

Reporter on insert“traps” readout ofgene expressionpattern


Mouse Mutagenesis

Program


Mouse Mutagenesis

Program

Key factors – HTP targeting

Mouse genome sequence•Reference sequence•Genomic informatic infrastructure•Indexed BAC libraries (129, B6)

BAC recombineering••Based on homologousBased on homologousrecombination in E. Colirecombination in E. Coli••Exploits genome sequenceExploits genome sequence

High throughput methods••SequencingSequencing••RoboticsRobotics


Mouse Mutagenesis

Program

Targeting Vector Construction PipelineTargeting Vector Construction Pipeline

Indexed BAC clonesIndexed BAC clones Plasmid sizedPlasmid sizedConditional Targeting VectorsConditional Targeting Vectors

96 Well96 WellRecombineeringRecombineering

KanRori

B1 B2

B3 B4

gal neopASA

PGKDTApA

AsiSIsite

KEY FEATURES:KEY FEATURES:••96 well format96 well format••Serial liquid transferSerial liquid transfer••Gateway based modularityGateway based modularity••Compatibility with indexed BAC resourcesCompatibility with indexed BAC resources


Mouse Mutagenesis

ProgramHigh Throughput Gene Targeting:

Conditional allele

lacZ-tagged allele (‘knockout first’*)

1 2gal::neo 3

FRTFRT

loxPloxP

*based on Testa et al., Genesis (2004)


Mouse Mutagenesis

Program

High Throughput Gene Targeting:Targeted trapping cassettes

E G R G S L L T C G D V E E N P G P

attL2

E G R G S L L T C G D V E E N P G P

attL1

T2A peptide*

T2A peptide

FRTgal neo pA

FRT

En2 SA

loxPframes 0 ,1, 2 and +ATG

pL1/L2GTØ,1,2 and K

*Szymczak et al., Nature Biotechnol. (2004)


Mouse Mutagenesis

Program

(1)Automated vector design software

~1 kb

~5 (3) kb ~5 kb

homology arms

phase2 1 1 0 0 0 0 0 02

intron size>0.5 kb >0.5 kb

deletion size

LacZ-NEO_loxP loxP

Design criteria:1) Minimal deletion of a single exon (flanking introns >800* bp or at least >500 bp)2) Cause frameshift and induce NMD3) Avoid deletion of conserved non-coding elements (>80%* identity to human)4) Select 70-mers (currently –50mers) for recombineering using ‘ArrayOligoSelector’

a. >300* bp upstream of splice acceptorb. >100* bp downstream of splice donorc. ~5* kb homology armsd. 5’ homology arm does not extend into promoter (required fortargeted trapping)


Mouse Mutagenesis

Program

Current EUCOMM gene targeting vector:


Mouse Mutagenesis

Program

» Automated vector design coupled to manual annotation

» 96-well vector construction pipeline (129, B6 BACs)

» Quality control of targeting vectors

» Small-scale electroporation of ES cells (129, B6)

» High efficiency targeted trapping of non-secreted proteins

High Throughput Gene Targeting:Key Achievements in Year 1

Sufficiently robust for high throughput production


Mouse Mutagenesis

Program

EUCOMM Gene list

8043 genes selected for targeted trapping

5173 Annotation pending

2870 Annotation Complete

71 Fail annotation (Assembly, Pseudogenes)

1004 Pending design

1038 Vector designs

757 Conditional design not possible


Mouse Mutagenesis

Program

EUCOMM Gene list

EUCOMM genes: >8000» 8043 selected

» 37 external requests

» 445 internal EUCOMM requests

Available on EUCOMM website (www.eucomm.org)» Status report

» Links to genome browser (DAS track)

» Links to gene information

» Searchable


Mouse Mutagenesis

Program


Mouse Mutagenesis

Program

(1)View vector designs on Ensembl genome browser


Mouse Mutagenesis

Program

Preparation of final vector plasmid DNAfor electroporation

Raw E-gel Image Processed E-gel Image

3-4µg plasmid DNAdigested with AsiS1

Plasmid DNAisolated using theQIAprep 96 Turbo

Miniprep kit(Qiagen)

Linearisedplasmid DNA

Linearised plasmid purified by ethanol precipitation


Mouse Mutagenesis

Program

Mutant ES cell production

BTX® HT 96/25 Well Electroporation System

1-2µg linearised final vector construct107 ggggb


Mouse Mutagenesis

Program

1/52/5

2/5Back up

Master

X5 copies

Internal andExternal archive

Genotyping and Sequencing

Genotyping and Sequencing

24 – 96 clonesselected

5 clonesselected

GenomicDNA isolation

(~1µg)

X-Gal staining to confirm positives

Matrix vials

ES clone selection


Mouse Mutagenesis

Program

• Designed to incubate, split and freeze

4000 gene trap clones a week

• Robot assembly consisting of:

- 2 Hamilton pipetting robots

- Cytomat automated incubator

- Robot arm

- Plate scanner

(for synchronization of clones)

• Currently test runs with cells

Automated GSF ES cell facility: Hamilton Cell Culture Robot


Mouse Mutagenesis

Program

InVitrogen E-gel

Processed

image

Cideagr1

Brd7gr1

Cideagr2

Brd7gr2

Cideagr1

Smarca5gr1

Brd7gr1

Cideagr2

Smarca5gr2

Brd7gr2

Standard reaction for all primers

attL2attL1FRT

gal neo pA

FRT

En2 SA

loxP loxP

gr1LF5

5-6kb (2 reactions)3’ arm genotyping


Mouse Mutagenesis

Program

Present status of targeting effort:

Gene targeting gene list: 8,488

Manual annotation: 3,194

Vector design: 906

Vector production: 378

Mutant ES cells: 98


Mouse Mutagenesis

Program

Introduction of null mutations

Conditional/ somatic mutations

Point mutations

Ectopic expression of genes

» any gene of interest can be integrated into EUCOMMvectors (gain-of-function studies, suicide genes,reporter genes, recombinases, siRNAs, etc.)

Mouse genetic tool kit to model human disease


Mouse Mutagenesis

Program

Priorization of conditional gene targeting:

EUCOMM asks for requests from the scientific community» [email protected]

» name,

» Ensembl ID of genes,

» brief statement about importance of this gene for your research,

» your name and contact address.


Mouse Mutagenesis

Program

• Identify loci with novel/useful

expression patterns

• 20 new conditional/inducible Cre

mouse strains (knock-in, Tg)

• Consolidate annotation/curation of

existing Cre databases, coordinate

new Cre driver strains

• Collect/deposit Cre driver mice in

European Mutant Mouse Archive

EUCOMM Cre Zoo and database

geneX del

Cre

geneX cond

Promoter Yy Cre

Gene X is inactivatedby Cre ONLY in tissue Y

X


Mouse Mutagenesis

Program

Cre

C EA/B

LBD

Cre

Cre recombinase

Human estrogen receptor a

Chimeric Cre-ERT2

recombinase E

Cre

ERT2

Cre

*Tam (*)

No ligand or E2

X

ERT2

LoxP LoxP


Mouse Mutagenesis

Program

Make use of the ES cell gene trap project to establish, by recombinase-mediatedcassette exchange (RMCE) in ES cells, mouse lines that cell-specifically expressCre-ERT2.

1 2SA pAgeo

LoxPLox5171

LoxP

Lox5171

SA pACre-ERT2

LoxP Lox5171Cre

1 SA pACre-ERT2

LoxP Lox5171

2

Initial Plan for Cre-ERT2 line establishment


Mouse Mutagenesis

Program

Validated Cre-ERT Lines (IGBMC&ICS)


Mouse Mutagenesis

Program

http://www.eucomm.org

general information(projects, participants,gene list, training,management, pressreleases, technology)

The automated mouse: Systematic generation of disease models

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