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Module 3: Knockout Resources Barry Rosen WTSI

Module 3: Knockout Resources Barry Rosen WTSI. Overview of High Throughput Mutant Mouse Vector and ES Cell Resources Survey of IKMC Alleles Brief Overview of EUCOMM/KOMP pipeline IKMC Database Interface EUCOMMTOOLS Alleles, Modular Vectors and Cre Alleles. OBJECTIVE:.

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Module 3: Knockout Resources Barry Rosen WTSI

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  1. Module 3: Knockout ResourcesBarry Rosen WTSI • Overview of High Throughput Mutant Mouse Vector and ES Cell Resources • Survey of IKMC Alleles • Brief Overview of EUCOMM/KOMP pipeline • IKMC Database Interface • EUCOMMTOOLS Alleles, Modular Vectors and Cre Alleles

  2. OBJECTIVE: -Increase Accessibility and Use of Mouse Models Through the Creation of a Comprehensive Resource of Modular Vector Resources and Gene Targeted ES Cells -Shift Emphasis from Vector/Targeted ES Cell Production to Phenotyping Mutants (in both Mice and ES Cells)

  3. IKMC Consolidates Mutant Mouse Resources

  4. International KO programs 21,000 genes IKMC Partners gene trapping gene targeting EUCOMM EUTOOLS KOMP NorCOMM mirKO TIGM Mutant ES cell resource lacZ-tagged null C57BL/6 Mutant mice Phenotyping programs International Mouse Knockout Consortium. (2007). Cell 128:9-13.

  5. KOMP EUCOMM/KOMP CSD Products: A dual resource of targeted ES cells and modular targeting constructs 8,000 genes (EUCOMM) 5,000 genes (KOMP CSD) 3,000 EUCOMMTOOLS (new program) 16,000 genes LacZ-tagged mutant ES cell resource Modular targeting constructs & cassettes EUCOMM KOMP mutant mice additional alleles Production Considerations versus User Considerations

  6. Common web portal for KO resourceswww.knockoutmouse.org

  7. International Mouse Phenotyping Consortium (IMPC) • Mouse models for the elucidation of mammalian gene function, insights into human biology and disease mechanisms • Production of thousands of mutant mice from IKMC ES cell resource • Systematic broad-based phenotyping • Common data archive and web portal • www.mousephenotype.org

  8. IKMC PROGRESS June 2012 http://www.knockoutmouse.org

  9. IKMC Progress to Goals

  10. IKMC PROGRESS

  11. IKMC PROGRESS

  12. Large Scale Mouse KO Projects

  13. KOMP CSD vs. RGN targeting(798 genes) 98% of genes targeted

  14. IKMC alleles Conditional Deletion ````` Random Gene Traps Targeted Non-Conditional http://www.knockoutmouse.org/about/

  15. Principle of Random Gene Trapping

  16. A public library of gene trap ES cells NIH-funded (1999 – 2004) WT- funded (2003 – 2005) www.genetrap.org

  17. International Gene Trap Consortium(IGTC) Website http://www.genetrap.org/

  18. Factors Enabling high-throughput gene targeting • Mouse genome sequence • Reference genome sequence (C57Bl/6) • Manual/automatic annotation of gene structures • Informatics of the genomic infrastructure • BAC recombineering • Homologous recombination in E. Coli • Indexed BAC libraries • Manipulate genomic clones with • nucleotide precision • High throughput methods • Sequencing • Robotics

  19. KOMP Regeneron Deletion Allele

  20. EUCOMM/KOMP(CSD) Conditional Alleles

  21. CE critical exons Designing: Identification of Critical Exon(s) phase 1 0 0 0 0 2 2 1 0 0 5’-most exon common to all mRNA isoforms which when deleted induces a frameshift mutation. NMD(nonsense mediated decay) should degrade mRNA

  22. Knockout-first allele: Promoterless selection cassette Type of Allele/State of “Critical Exon” RN/TT tm1a FRT loxP Conditional Ready 1 lacZ neo 2 3 Flp Cre WT/C RN/D tm1b tm1c lacZ neo 1 3 1 2 3 RN: Reporter Null TT: Targeted Trap C: Conditional N: Null D: Deletion Cre N/D tm1d 1 3

  23. Knockout-first allele with Promoter RN/TT+Pr tm1a FRT FRT loxP loxP Conditional Ready loxP 1 lacZ neo 2 3 WT/C Flp Cre RN/D tm1b tm1c 1 2 3 lacZ 1 3 RN: Reporter Null TT: Targeted Trap C: Conditional N: Null D: Deletion +P: Contains Promoter Cre N/D tm1d 1 3

  24. Knockout-first allele* EUCOMM/KOMP-CSD 40% clones 40% clones *based on Testa et.al, Genesis, 2004

  25. Conditional targeting strategy for single exon genes-Artificial Intron Artificial Intron

  26. IKMC ALLELE POTENTIALS =Frt SSR Exposure Flp Cre Conditional Potential =LoxP Pipeline None Flp Cre Deletion (lacZ replacement): ATG - - no KOMP-REGN RN/D+Pr RN/D 1 lacZ neo pA pA EUCOMM KOMP-CSD yes RN/TT RN/D WT/C N/D CE CE Targeted, non-conditional (promoterless) Deletion (lacZ-tagged) Targeted, non-conditional (promoter) EUCOMM KOMP-CSD yes RN/TT+Pr RN/D WT/C N/D CE 1 1 lacZ neo 2 1 3 neo 2 lacZ lacZ neo 3 3 SA pA pA SA pA pA pA SA N/D RN/D - RN/D+Pr no KOMP-CSD EUCOMM KOMP-CSD no WT/Rev RN/TT - RN/TT+Pr CE EUCOMM KOMP-CSD WT/Rev - no RN/TT - CE Knockout-first (promoter) 1 lacZ neo 2 3 SA pA pA WT allele Knockout-first (promoterless) 1 1 lacZ neo 2 2 3 3 pA SA

  27. June 2011

  28. High Throughput Targeting Pipeline I) In silicoPhase II)Vector Construction Phase III)ES cell Gene Targeting Phase

  29. Gene Annotation and Design • Design on automatically annotated genes risky • Original designs were on genes with full manual annotation • Slow, becomes a bottleneck! • “Design First” Strategy • Automated scripts run on genome(Vega/Ensembl) • Confirmation of design by annotator • Higher througput, >95% accuracy

  30. CE LacZ_neo_pA loxP Gateway Cassette >0.1kb 0.3kb 0.1kb >0.3kb G5 U5 U3 D5 D3 G3 intron size ~1 kb >0.7 kb >0.7 kb homology arms ~5 kb ~5 kb Automatic Selection of Recombineering oligos Oligo postions define a DESIGN BAC clone deletion size - pre-defined distances from CE - candidate regions (U, D and G) - 50mers - unique hit on the BAC (100kb either site of CE) -avoid deletion of conserved elements ARRAY OLIGO SELECTOR PROGRAM(AOS)

  31. View vector designs on genome browser

  32. 96-well serial recombineering

  33. 3-way Gateway reaction L3L4_DTA

  34. C57BL/6N ES cells JM8.F6 JM8.N4 JM8 GIBCO KO medium 10%-15% serum + Glutamine + BME + LIF Kent Lloyd UC Davis feeder-free SNL feeders JM8.F6 SNP panel CGH JM8.N4 1.7 Mb gain Chr 10 Pettit et al., 2009, Nature Methods

  35. C57BL/6N ES cell validation: Establishment of a JM8 Agouti cell line *GLT, germline transmission Non-agouti (a) A B C D 2 3 4 VL30 Testcross: JM8.F6 (A/a) chimera C57BL/6N (a/a) C57BL/6N (A/a) X Pettitt S. Et al., 2009, Nature Methods

  36. Genotyping Strategy: LRPCR-Seq 3’ homology arm 5’ homology arm FRT FRTloxP loxP 1 lacZ neo 2 3 LR-PCR primers GR GF 5’U 3’U LX cassette LR-PCR products 3’ arm 5’ arm Sequencing primers GF GR 5’Us 3’Us LR Skarnes et.al., 2011 Nature

  37. Loss of Allele Genotyping(LOA) Can Also be Employed

  38. WHAT HAVE WE LEARNED ABOUT GENE TARGETING? • Gene Targeting Can Be Highly Efficient • 40% Average Targeting Frequency • 35% Using Promoter Driven Selection • >70% Using Promoterless Selection • Gene Restrictions for Promoterless Vectors • Factors Contributing to High Targeting Frequencies: • Long(10 kb total) homology arms • Virtually isogenic DNA • Strong Negative Selection(DTA) • Optimal Cell Handling

  39. ALL GENES CAN BE TARGETED, MOST AT HIGH FREQUENCY 80% of time, re-prepping vector and repeating electroporation rescues failed project >50% of time, re-designing vector around new critical exon|(s) rescues failed project

  40. Common web portal for KO resourceswww.knockoutmouse.org

  41. Looking up a gene

  42. Visualization of Vector Maps Genbank File Synthetic Vector Map

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