The automated mouse: Systematic generation of disease models

Transcription

1 The automated mouse: Systematic generation of disease models Nadia Rosenthal EMBL Mouse Biology Monterotondo, Rome Harefield Heart Science Centre Imperial College London Australian Regenerative Medicine Institute Monash University, Melbourne

2 European mouse Mouse mutagenesis Mutagenesis and phenotyping programs 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

3 Gene knockout vs. conditional mutatagenesis Knockout Conditional knockout X genex Promoter Y y Cre genex cond Targeted deletion of critical exon Cre Cre-mediated excision of critical exon genex del Gene X is inactivated in all tissues genex del Gene X is inactivated by Cre ONLY in tissue Y

4 EUCOMM Objectives:» Generation of up to 12,000 conditional gene trap mutations in ES cells» 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)

5 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

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7 Wellcome Trust Sanger Institute A. Bradley (coordinator), W. Skarnes, P. Liu Medical Research Council S. Brown Max-Planck-Institute of Molecular Genetics P. Ruiz University of Dresden F. Stewart GSF National Research Center W. Wurst (coordinator), M. Hrabé de Angelis Institute Clinique de la Souris (ICS) J.Auwerx, P. Chambon Deutsches Ressourcenzentrum für Genomforschung (RZPD) A. Hörlein University of Frankfurt H. von Melchner EMBL N. Rosenthal National Research Council G. Tocchini-Valentini

8 Structure of EUCOMM Subprojects (SP) and Work Packages (WP)

9 A dual resource of targeted ES cells and modular targeting constructs 13,000 genes 8,000 genes (EUCOMM) 5,000 genes (KOMP) EUCOMM KOMP LacZ-tagged conditional mutant ES cell resource Modular targeting constructs & cassettes mutant mice additional alleles

10 Gene target vs. trap Target Trap genex genex Targeted deletion of critical exon Insertion of a new coding region, disrupting gene genex del Gene X is inactivated in all tissues Reporter on insert traps readout of gene expression pattern

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12 Conditional gene trap vectors Mouse Mutagenesis rfliprosa geo frt F3 frt F3 geo pa LTR loxp lox5171 LTR SA loxp lox5171 rfliprosaceo frt F3 SA TM SA loxp lox5171 Ceo pa LTR frt F3 loxp lox5171 Schnütgen et al, PNAS, 102, 7221, 2005 LTR

13 Ex 1 SA geo pa Ex 2 mutation + FLPe SA geo pa Ex 1 Ex 2 no mutation + Cre Ex 1 SA geo pa Ex 2 mutation

14 Validation of conditional gene trap vector in vitro t SA geo pa E1 E3 E5 + FLPe + FLPe wt inv endo t inv + Cre wt re-inv fus endo fus endo fus endo fus RBBP7 lamin A re-inv wt pol II t inv re-inv t inv + Cre re-inv

15 Present status of gene trap:» Production of 44,800» Generation of 22,000 clones» Sequence of 2,000 1,212 clones successful processed representing approximately 700 different genes» Success rate of sequencing and annotation of products is approximately 63%» Present weekly production rate: 4,000 clones per week

16 Gene target vs. trap Target Trap genex genex Targeted deletion of critical exon genex del Gene X is inactivated in all tissues Insertion of a new coding region, disrupting gene Reporter on insert traps readout of gene expression pattern

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18 Key factors HTP targeting Mouse genome sequence Reference sequence Genomic informatic infrastructure Indexed BAC libraries (129, B6) BAC recombineering Based on homologous recombination in E. Coli Exploits genome sequence High throughput methods Sequencing Robotics

19 Targeting Vector Construction Pipeline Plasmid sized Conditional Targeting Vectors Indexed BAC clones B1 96 Well Recombineering SA gal neopa B3 ori KanR B2 B4 pa DTA PGK AsiSI site KEY FEATURES: 96 well format Serial liquid transfer Gateway based modularity Compatibility with indexed BAC resources

20 High Throughput Gene Targeting: Conditional allele lacz-tagged allele ( knockout first *) FRT 1 FRT gal::neo 2 loxp 3 loxp *based on Testa et al., Genesis (2004)

21 High Throughput Gene Targeting: trapping cassettes Targeted T2A peptide* EGRGSLLTCGDVEENPGP attl1 FRT En2 SA gal FR neo pat attl2 pl1/l2gtø,1,2 and K loxp frames 0,1, 2 and +ATG EGRGSLLTCGDVEENPGP T2A peptide *Szymczak et al., Nature Biotechnol. (2004)

22 (1)Automated vector design software LacZ-NEO_loxP 2 loxp >0.5 kb phase intron size >0.5 kb ~1 kb deletion size homology arms ~5 (3) kb ~5 kb Design criteria: 1) Minimal deletion of a single exon (flanking introns >800* bp or at least >500 bp) 2) Cause frameshift and induce NMD 3) 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 acceptor b. >100* bp downstream of splice donor c. ~5* kb homology arms d. 5 homology arm does not extend into promoter (required for targeted trapping)

23 Current EUCOMM gene targeting vector:

24 High Throughput Gene Targeting: Key Achievements in Year 1»»»»» 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 Sufficiently robust for high throughput production

25 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

26 EUCOMM Gene list EUCOMM genes: >8000»»» 8043 selected 37 external requests 445 internal EUCOMM requests Available on EUCOMM website ( Status report Links to genome browser (DAS track) Links to gene information Searchable

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28 (1)View vector designs on Ensembl genome browser

29 Preparation of final vector plasmid DNA for electroporation Raw E-gel Image Processed E-gel Image Plasmid DNA isolated using the QIAprep 96 Turbo Miniprep kit (Qiagen) 3-4µg plasmid DNA digested with AsiS1 Linearised plasmid DNA Linearised plasmid purified by ethanol precipitation

30 Mutant ES cell production 1-2µg linearised final vector construct 107 ggggb BTX HT 96/25 Well Electroporation System

31 ES clone selection X-Gal staining to confirm positives 2/ clones selected Back up 2/5 1/5 Genomic DNA isolation (~1µg) Matrix vials X5 copies Genotyping and Sequencing Internal and External archive 5 clones selected Master Genotyping and Sequencing

32 Automated GSF ES cell facility: Hamilton Cell Culture Robot 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

33 3 arm genotyping 5-6kb (2 reactions) LF5 attl1 attl2 FRT loxp FRT En2 SA gal neo gr1 loxp pa InVitrogen E-gel Processed image Standard reaction for all primers Cidea gr1 Brd7 Cidea gr1 gr2 Brd7 gr2 Cidea Brd7 Cidea Brd7 gr1 gr1 gr2 gr2 Smarca5 Smarca5 gr2 gr1

34 Present status of targeting effort: Gene targeting gene list: Manual annotation: Vector design: Vector production: Mutant ES cells: 8,488 3,

35 Mouse genetic tool kit to model human disease Introduction of null mutations Conditional/ somatic mutations Point mutations Ectopic expression of genes» any gene of interest can be integrated into EUCOMM vectors (gain-of-function studies, suicide genes, reporter genes, recombinases, sirnas, etc.)

36 Priorization of conditional gene targeting: EUCOMM asks for requests from the scientific community»»»»» name, Ensembl ID of genes, brief statement about importance of this gene for your research, your name and contact address.

37 EUCOMM Cre Zoo and database X Identify loci with novel/useful Promoter Y expression patterns y Cre 20 new conditional/inducible Cre mouse strains (knock-in, Tg) Consolidate annotation/curation of Cre existing Cre databases, coordinate new Cre driver strains genex del Collect/deposit Cre driver mice in European Mutant Mouse Archive genex cond Gene X is inactivated by Cre ONLY in tissue Y

38 Cre recombinase Cre Human estrogen receptor a A/B Chimeric Cre-ERT2 recombinase Cre ERT2 Cre No ligand or E2 LoxP LoxP Tam (*) Cre *T2 ER C E LBD E X

39 Initial Plan for Cre-ERT2 line establishment Lox5171 LoxP 1 SA Lox5171 LoxP geo Cre LoxP SA 1 SA Lox5171 Cre-ERT2 pa Cre-ERT2 Lox5171 pa LoxP 2 pa 2 Make use of the ES cell gene trap project to establish, by recombinase-mediated cassette exchange (RMCE) in ES cells, mouse lines that cell-specifically express Cre-ERT2.

40 Validated Cre-ERT Lines (IGBMC&ICS)

41 general information (projects, participants, gene list, training, management, press releases, technology)