the automated mouse: systematic generation of disease models
TRANSCRIPT
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
European Conditional
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
European Conditional
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:
European Conditional
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
European Conditional
Mouse Mutagenesis
Program
European Conditional
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
European Conditional
Mouse Mutagenesis
Program
Subprojects (SP) and Work Packages (WP)
Structure of EUCOMM
European Conditional
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
European Conditional
Mouse Mutagenesis
ProgramGene target vs. trap
geneX
geneX del
Gene X is inactivatedin all tissues
geneX
Targeted deletionof critical exon
Insertion of a new codingregion, disrupting gene
Target Trap
Reporter on insert“traps” readout ofgene expressionpattern
European Conditional
Mouse Mutagenesis
Program
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
Mouse Mutagenesis
ProgramGene target vs. trap
geneX
geneX del
Gene X is inactivatedin all tissues
geneX
Targeted deletionof critical exon
Insertion of a new codingregion, disrupting gene
Target Trap
Reporter on insert“traps” readout ofgene expressionpattern
European Conditional
Mouse Mutagenesis
Program
European Conditional
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
European Conditional
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
European Conditional
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)
European Conditional
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)
European Conditional
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)
European Conditional
Mouse Mutagenesis
Program
Current EUCOMM gene targeting vector:
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
Mouse Mutagenesis
Program
European Conditional
Mouse Mutagenesis
Program
(1)View vector designs on Ensembl genome browser
European Conditional
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
European Conditional
Mouse Mutagenesis
Program
Mutant ES cell production
BTX® HT 96/25 Well Electroporation System
1-2µg linearised final vector construct107 ggggb
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
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.
European Conditional
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
European Conditional
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
European Conditional
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
European Conditional
Mouse Mutagenesis
Program
Validated Cre-ERT Lines (IGBMC&ICS)
European Conditional
Mouse Mutagenesis
Program
http://www.eucomm.org
general information(projects, participants,gene list, training,management, pressreleases, technology)