Knockout Mouse Project (KOMP) and Knockout Mouse Production and

Phenotyping (KOMP2)

Mouse 101Oct 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.

1 2bgal 3

lacZ-tagged allele (‘knockout first’*)

FRTFRT

loxPloxP

EU/KOMP multi-purpose allele

*based on Testa et al., Genesis, 2004

1 2bgal 3

lacZ-tagged allele (‘knockout first’*)

FRTFRT

loxPloxP

EU/KOMP multi-purpose allele

*based on Testa et al., Genesis, 2004

lacZ-tagged null allele ( D exon)

1 bgal 3

FRTFRT

loxP

Cre recombinase

1 2bgal 3

lacZ-tagged allele (‘knockout first’*)

FRTFRT

loxPloxP

EU/KOMP multi-purpose allele

*based on Testa et al., Genesis, 2004

pre-conditional allele (wild-type)

1 2 3

null allele (D exon, frameshift, NMD)

Flp recombinase

Cre recombinase

1 3

Regeneron - Velocigene

Target Gene

ATG TGA

deletion

loxP

lacZ–pA

loxP

hUbCpro-neor–pA

Insert reporter/selection cassetteby recombineering

Screen ES cells by loss of allelequantitative PCR assay

KOMP - Goals and Progress

Date

1/1/

07

3/1/

07

5/1/

07

7/1/

07

9/1/

07

11/1

/07

1/1/

08

3/1/

08

5/1/

08

7/1/

08

9/1/

08

11/1

/08

1/1/

09

3/1/

09

5/1/

09

7/1/

09

9/1/

09

11/1

/09

1/1/

10

3/1/

10

5/1/

10

7/1/

10

9/1/

10

11/1

/10

1/1/

11

3/1/

11

5/1/

11

7/1/

11

9/1/

11

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

KOMP ProductionMutant ES cell goals Mutant ES cell production

9090 KO lines produced and being distributed from the KOMP repository

Community uptake:1250 orders for vectors2512 orders for ES cells980 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 toolsNature 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 GenesWhite et al., CELL 154, 452-464, 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 phenotype245 (52%) strains had 2-5 phenotypes

Andy Peterson, Genentech

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

KOMP2 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

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

StrengthBody 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 100x Fbxo7-/- bone marrow 100x

WT spleen 40x Fbxo7-/- spleen 40x

RBCs CD3+ T cells

OPT

Embryonic Lethal Pipeline

Embryo viability checkpoint

μCT

Genotype pups(P14-21)HET × HET

Embryo Assessment

0% HOM

E1

2.5

E15.5 HOME9.5 HOM

+ Targeted HistopathologyEmbryo & Placenta

HET

Adult HET Phenotyping

TCF/Lef-LacZ reporter

+ Targeted HistopathologyEmbryo & 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)

WT Satb2 -\-

2mm

Satb2 – uCT Imaging

Klhdc2: embryonic lethal with multiple defects

homhet

homhet

homhet

homhet

Database Access

www.mousephenotype.org

Korean Mouse Phenotyping Centre