Post on 18-Jan-2016
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