Knockout Mouse Project (KOMP) and Knockout Mouse Production and Phenotyping (KOMP 2 ) Mouse 101 Oct 19, 2015

The vision for KOMP was articulated in a meeting at the Banbury Center, Cold Spring Harbor in 2003, calling for high throughput production of gene knockouts, and phenotyping, for every gene in the mouse genome.

Deltagen/Lexicon Lines

KOMP ( ) “…a high-throughput international effort to produce…knockouts for all mouse genes, and place these resources into the public domain.” The KOMP was launched in 2006 by NIH – $56.6 million over 5 years from the ICs – a goal of creating 8,500 ES cell lines – alleles are nulls or conditional-ready, contain reporter The EC launched EUCOMM, the European Conditional Mouse Mutagenesis Program in October 2005 (funded in Feb 2005) – 13 M Euros over 3 years – a goal of creating 8,000 mutants. KOMP and EUCOMM along with other international efforts formed the International Knockout Mouse Consortium (IKMC) and have jointly produced > 17,000 mutant ES cell lines and made them available from public repositories.

12  gal 3 lacZ-tagged allele (‘knockout first’*) FRT loxP EU/KOMP multi-purpose allele *based on Testa et al., Genesis, 2004

12  gal 3 lacZ-tagged allele (‘knockout first’*) FRT loxP EU/KOMP multi-purpose allele *based on Testa et al., Genesis, 2004 lacZ-tagged null allele (  exon) 1  gal 3 FRT loxP Cre recombinase

12  gal 3 lacZ-tagged allele (‘knockout first’*) FRT loxP EU/KOMP multi-purpose allele *based on Testa et al., Genesis, 2004 pre-conditional allele (wild-type) 123 null allele (  exon, frameshift, NMD) Flp recombinase Cre recombinase 13

Regeneron - Velocigene Target Gene ATG TGA deletion loxP lacZ–pA loxP hUbCpro-neo r –pA Insert reporter/selection cassette by recombineering Screen ES cells by loss of allele quantitative PCR assay

KOMP - Goals and Progress 9090 KO lines produced and being distributed from the KOMP repository Community uptake: 1250 orders for vectors 2512 orders for ES cells 980 orders for mice or germplasm

KOMP2 - Scientific Rationale Provides access to unannotated genes by providing hypothesis testing and tools Provides new insights into pleiotropy

KOMP2 - Scientific Rationale Provides access to unannotated genes by providing hypothesis testing and tools Nature Commentary

KOMP2 - Scientific Rationale Provides access to unannotated genes by providing hypothesis testing and tools Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes White et al., CELL 154, , July 2013

KOMP2 - Scientific Rationale Provides access to unannotated genes by providing hypothesis testing and tools Provides new insights into pleiotropy 472 Mouse knockouts were broadly phenotyped 130 (27%) strains had 1 phenotype 245 (52%) strains had 2-5 phenotypes Andy Peterson, Genentech

KOMP2 - Policy Rationale KOMP2 - Policy Rationale Eliminates duplication and waste Sets the standard for reproducibility Includes sex as a biological variable

KOMP2 Project Goals - ( ) Phase 1 ( ): Phenotype up to 2,500 lines – Pipeline development, logistics – Phenotype technology developments – Economies of scale Phase 2 ( ): Phenotype 6,000 mutants – Business plan in preparation Data freely available through a Data Coordination Center – “One stop shop” Web Portal Mice available through the global network of mouse repositories Coordinate with IMPC to achieve broad-based phenotyping of 20,000 mutants from the IKMC resource – A collaborative activity of mouse centers worldwide

KOMP2 - Goals and Progress

General Immune Musculo- skeletal Sensory Pulmonary Cardiovascular Metabolism Neurological/ Behaviour Modified SHIRPA/Dysmorphology Grip Strength ECG / Echo Intraperitoneal Glucose Tolerance Test Auditory Brain Stem Response (2+2) Body Composition (DEXA) X-ray (5 + 5) Slit Lamp Opthalmoscope Hematology Clinical Blood Chemistry Insulin Blood Level Gross Pathology & Tissue Collection (2+2) Tissue embedding &Block Banking (2+2) Heart Weight Calorimetry Challenge Whole Body Plethysmography Acoustic Startle/PPI Histopathology (2+2) - from blocks where required FACS analysis – blood/spleen Open Field Pain Test Grip Strength Body Composition (DEXA) Modified SHIRPA/Dysmorphology Weight Reproduction Fertility Multiple Physiological Domains

Phenotyping Pipeline Embryo In Vivo Terminal

Fbxo7 Phenotyping MGI GO biological process: negative regulation of lymphocyte differentiation IMPC phenotype: CBC, clinical blood chemistry, male infertility WT bone marrow 100xFbxo7 -/- bone marrow 100x WT spleen 40xFbxo7 -/- spleen 40x RBCs CD3+ T cells

OPT Embryonic Lethal Pipeline Embryo viability checkpoint μCT Genotype pups (P14-21) HET × HET Embryo Assessment 0% HOM E12.5 E15.5 HOME9.5 HOM + Targeted Histopathology Embryo & Placenta HET Adult HET Phenotyping TCF/Lef-LacZ reporter + Targeted Histopathology Embryo & Placenta HET × HET LacZ

Tmem100 - Transmembrane Protein 100 Functions downstream of the BMP/ALK1 signaling pathway HOM lethal at E12.5 – lacZ staining found predominantly in arterial endothelial cells and heart (arrow) HOM viable at E9.5 – HOM embryos have large pericardial effusion (arrow) and cardiac dsymorphology and enlargement as seen in both brightfield microscopy and OPT (arrow) -/- E9.5 +/+ E9.5 -/- E9.5 OPT BF +/- E12.5 lacZ Tmem100 – OPT Imaging

Michael Wong and Mark Henkelman, Mouse Imaging Centre Satb2 - special AT-rich sequence binding protein 2 Satb2 -/- found dead at P0/P1 (n=9) E15.5 microCT analysis – population average (n=8) WT and Satb2 -\- Visually evident phenotypes: missing palate, shorter tongue and mandible Volumetric analysis: much smaller tongue and mandible (FDR threshold of 5%) (blue indicates structures that are smaller than WT and pink structures that are larger than WT) WTSatb2 -\- 2mm Satb2 – uCT Imaging

Klhdc2: embryonic lethal with multiple defects homhet homhet homhet homhet

Database Access

Korean Mouse Phenotyping Centre