Knockout Mouse Project KOMP and Knockout Mouse Production

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 http: //www. informatics. jax. org/external/ko/

KOMP (2006 -2011) • “…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.

EU/KOMP multi-purpose allele lac. Z-tagged allele (‘knockout first’*) FRT 1 FRT bgal 2 lox. P 3 lox. P *based on Testa et al. , Genesis, 2004

EU/KOMP multi-purpose allele lac. Z-tagged allele (‘knockout first’*) FRT 1 FRT bgal 2 lox. P 3 lox. P Cre recombinase FRT 1 FRT bgal 3 lox. P lac. Z-tagged null allele (D exon) *based on Testa et al. , Genesis, 2004

EU/KOMP multi-purpose allele lac. Z-tagged allele (‘knockout first’*) FRT bgal 1 2 lox. P 3 lox. P Flp recombinase 1 2 3 pre-conditional allele (wild-type) Cre recombinase 1 3 null allele (D exon, frameshift, NMD) *based on Testa et al. , Genesis, 2004

Regeneron - Velocigene Screen ES cells by loss of allele quantitative PCR assay Insert reporter/selection cassette by recombineering ATG TGA Target Gene deletion lac. Z–p. A lox. P h. Ub. Cpro-neor–p. A

KOMP - Goals and Progress KOMP Production Mutant ES cell goals Mutant ES cell production 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 D 1/ ate 1/ 3/ 07 1/ 5/ 07 1/ 7/ 07 1/ 9/ 07 1 11 /0 /1 7 / 1/ 07 1/ 3/ 08 1/ 5/ 08 1/ 7/ 08 1/ 9/ 08 1 11 /0 /1 8 / 1/ 08 1/ 3/ 09 1/ 5/ 09 1/ 7/ 09 1/ 9/ 09 1 11 /0 /1 9 / 1/ 09 1/ 3/ 10 1/ 5/ 10 1/ 7/ 10 1/ 9/ 10 1 11 /1 /1 0 / 1/ 10 1/ 3/ 11 1/ 5/ 11 1/ 7/ 11 1/ 9/ 11 1/ 11 0 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

KOMP 2 - Scientific Rationale • Provides access to unannotated genes by providing hypothesis testing and tools • Provides new insights into pleiotropy

KOMP 2 - Scientific Rationale • Provides access to unannotated genes by providing hypothesis testing and tools Nature Commentary

KOMP 2 - 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, 452 -464, July 2013

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

KOMP 2 - Policy Rationale • Eliminates duplication and waste • Sets the standard for reproducibility • Includes sex as a biological variable

KOMP 2 Project Goals - (2011 -2021) • • Phase 1 (2011 -2016): Phenotype up to 2, 500 lines – Pipeline development, logistics – Phenotype technology developments – Economies of scale Phase 2 (2016 -2021): 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

KOMP 2 - Goals and Progress

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

Phenotyping Pipeline Embryo In Vivo Terminal

Fbxo 7 Phenotyping • MGI GO biological process: negative regulation of lymphocyte differentiation • IMPC phenotype: CBC, clinical blood chemistry, male infertility RBCs WT bone marrow 100 x Fbxo 7 -/- bone marrow 100 x WT spleen 40 x Fbxo 7 -/- spleen 40 x CD 3+ T cells

Embryonic Lethal Pipeline Adult HET Phenotyping HET × HET Genotype pups (P 14 -21) 0% HOM HET × HET Embryo viability checkpoint E 12. 5 Embryo Assessment OPT E 9. 5 HOM + Targeted Histopathology Embryo & Placenta Lac. Z TCF/Lef-Lac. Z reporter μCT E 15. 5 HOM + Targeted Histopathology Embryo & Placenta

Tmem 100 – OPT Imaging OPT Tmem 100 - Transmembrane Protein 100 +/+ E 9. 5 Functions downstream of the BMP/ALK 1 signaling pathway • HOM lethal at E 12. 5 – lac. Z staining found predominantly in arterial endothelial cells and heart (arrow) • HOM viable at E 9. 5 – HOM embryos have large pericardial effusion (arrow) and cardiac dsymorphology and enlargement as seen in both brightfield microscopy and OPT (arrow) -/- E 9. 5 lac. Z +/- E 12. 5 • BF -/- E 9. 5

Satb 2 – u. CT Imaging Satb 2 - special AT-rich sequence binding protein 2 • Satb 2 -/- found dead at P 0/P 1 (n=9) • • • E 15. 5 micro. CT analysis – population average (n=8) WT and Satb 2 -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) WT Satb 2 -- 2 mm Michael Wong and Mark Henkelman, Mouse Imaging Centre

Klhdc 2: embryonic lethal with multiple defects het hom

Database Access

www. mousephenotype. org

Korean Mouse Phenotyping Centre