Kent Lloyd KOMP Repository, MMRRC-UC Davis
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1 Kent Lloyd KOMP Repository, MMRRC-UC Davis
2 KOMP 2011 and beyond Nature Genetics 2005;36,
3 KOMP Repository THE KNOCKOUT MOUSE PROJECT and FRT attl1 SA FRT loxp βgal neo pa attl2 ZeoR attr1 PheS attr2 loxp microinjection 5 arm attr3 ori AmpR 3 arm attr4 mice ES cells cryopreservation/ cryorecovery sperm & embryos CmR ccdb PGK DTA pa attl3 attl4 ori KanR vectors Sponsored by (U42):
4 IKMC (INTERNATIONAL KNOCKOUT MOUSE CONSORTIUM) KOMP -C57BL6/N -lacz-tagged -K/O alleles: conditional null targeted trap deletion 17,000 genes Mutant ES cell resource 8,500 genes (KOMP) 8,000 genes (EUCOMM) 500 genes (NorCOMM) + Vector resource mutant mice/yr
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6 Efforts to solicit input: --online surveys --RFI --conferences 6
7 KOMP Phenotyping Survey September 8, October 15, 2009 ~300 respondents out of 2517 To ascertain what researchers would want to see from a primary phenotype screen. NO 97 YES 241 7
8 Survey Summary Report Question #2: Thinking beyond your laboratory, what do you see as the 3 essential tests, analyses, and/or examinations that would most likely reveal the utility of a mutant mouse line in your field? Two caveats: the numbers of mice used per test are limited to 5-10 and the tests must be high throughput (100 s/y). immunity/infection/inflammation/arthritis/a diabetes/metabolism/mitochondrial/endocri behavior/neurology/sleep histology/morphology hematology/facs/bleeding embryonic blood chemistry/lipid levels body composition/diet/growth reproduction/litter size/puberty gene expression bone density/bone morhology/bone strength EKG/cardiac defects/cardiovascular cancer proteomics/genotyping/gene retina/vision/ophthalmology imaging/microct lacz hypertension hearing muscle aging GI urinalysis genotoxic sensitivity/radiation biochemistry/cell biology respiratory dermatology/hair karyotype/stem cells oral/teeth olfactory exercise pharmacodynamics renal function vocalization wound healing secretory gland function tests thrombosis behavior immuno metabolism 8
9 Survey Summary Report Question #3: In general, should tests be administered under naïve and/or challenge conditions? Response: 271 responses; 182 (67%) left comments Neither, 5 Both, 193 Baseline, 67 Challenge, 6 9
10 KOMP Phenotyping RFI September 24, October 15, 2009 Issued by NCRR, NHGRI, & NICHD ~25 respondents (anonymous) To seek input on large scale, high-throughput phenotyping of KOMP mice.
11 RFI Summary Report Question #6: How would you value an NIH-subsidized program of standardized phenotypes in a high throughput manner, at a lower cost per gene and encompassing more fields than individual labs could perform? Response: 23 responses (92% of RFI respondents) all AGREED on value Would the availability of data (regardless of the outcome of the experiment) be of benefit to the larger community? Response: 23 responses (92% of RFI respondents) all AGREED on benefit Would this effort help individual PIs focus on custom phenotyping of fewer but very relevant genes? Response: 23 responses (92% of RFI respondents) all AGREED would help focus Approximately what range of costs does your lab invest in phenotyping, with or without challenges (please include all personnel and overhead cost, as well as mouse production)? Response: 12 responses (46% of RFI respondents) Costs per line: $15K - $175K (5 responses) Costs per lab: $100K - $3M (7 responses)
12 KOMP Phenotyping Conference October 29-30, 2009 Hyatt Regency Hotel, Bethesda, Maryland IKMC PI s, invited researchers, NIH staff 12
13 KOMP Phenotyping Conference Consensus: 1) Gene selection (directed, not encyclopedic ) 2) Include unannotated genes 3) Mouse production (convert ESC to mice, cost) 4) High throughput tests (baseline, informative) 5) Data access and availability (free, real-time) 6) Coordinate with international efforts 13
14 2011 and beyond IMPC (INTERNATIONAL MOUSE PHENOTYPING CONSORTIUM)
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16 IMPC Phenotyping Conference 16
17 IMPC Phenotyping Conference April 7-9, 2010 c Toronto Center for Phenogenomics, Canada IMPC participants, international funders, research/phenotyping scientists 17
18 IMPC Phenotyping Conference Highlights: 1) Imaging 2) Embryonic lethality 3) Planning informatics infrastructure 4) Industry perspectives
19 IMPC Phenotyping Conference IMAGING Take home messages: important, affordable, right thing to do data, functional, non-invasive, non-lethal quick conceptual analysis of complex structures (cf histology) MRI (brain & embryos other tissues?) CT (embryos; KI soaking resolution, scan time) OPT (for young adults) Mark Henkelman, The Hospital for Sick Children Jason Lerch, The Hospital for Sick Children
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23 IMPC Phenotyping Conference EMBRYONIC LETHALITY Take home messages: identify first by lack of homos in het crosses screen must consider placental & CVS phenotype cause of death related to gestational time of death homozygous lethal points to heterozygous pheno insight into human disease pathways identify druggable (human) targets imaging: non-invasive in utero echocardiography Janet Rossant, The Hospital for Sick Children Cecilia Lo, University of Pittsburgh ( bench to bassinette )
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26 IMPC Phenotyping Conference PLANNING INFORMATICS INFRASTRUCTURE Phenotyping effort will generate large amts data 2 components to informatics effort: data collection and export at production sites data center depot and portal at informatics hub Shared governance (phenotypers, funders) Issues and challenges consensus on data presentation data curation and qc images and other large data sets evaluation and continuing maintenance budget
27 IMPC Phenotyping Conference INDUSTRY PERSPECTIVES Nick Gale (Regeneron,Inc) lacz expression analysis informs phenotype molecular phenotyping (microarray) part of KOMP mutagenesis project Andy Peterson (Genentech, Inc) immunological, metabolic phenotypes common phenotype & expression pattern incongruent (may be due to extracellular proteins) deposited 496 mutant lines & data in MMRRC
28 Activities at UC Davis: KOMP mutagenesis (CSD Consortium) KOMP repository (with CHORI) KOMP phenotyping pilot (ARRA funding): Fertility & Viability (312) LacZ Tissue Dist (312) Necropsy (100) Illumina (100) Physical & Behavioral Exams (100) Demonstrate Capability (# TBD) Histology Embryonic Lethals Depends on Resources (~60) Frozen Sect & Wholemount Annotate Tissue Wts Obvious Pathology LacZ+ tissues ~4 M mice/gene ~5 Tissues Orphan Gene Emphasis Physical Exam Modified SHIRPA Serum Chem Histochem Behavior Metab Disease Imaging Other Database & Web Presence
29 Mouse Biology Program mousebiology.org nd Street Suite 400 Davis, CA (530) 754-MOUSE
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a) Stock 4: 252 flies total: 120 are P[w+]: 64 are CyO; 56 are TM3; all are females. 132 are w-: 68 are CyO; 64 are TM3; all are males.
![a) Stock 4: 252 flies total: 120 are P[w+]: 64 are CyO; 56 are TM3; all are females. 132 are w-: 68 are CyO; 64 are TM3; all are males.](/thumbs/91/106088469.jpg "a) Stock 4: 252 flies total: 120 are P[w+]: 64 are CyO; 56 are TM3; all are females. 132 are w-: 68 are CyO; 64 are TM3; all are males.")
Drosophila Problem Set 2018 1) You have created P element transformants of a construct that contains the mini-white gene, which confers an orange eye color in a homozygous white mutant background. For
(a) Scheme depicting the strategy used to generate the ko and conditional alleles. (b) RT-PCR for
Supplementary Figure 1 Generation of Diaph3 ko mice. (a) Scheme depicting the strategy used to generate the ko and conditional alleles. (b) RT-PCR for different regions of Diaph3 mrna from WT, heterozygote
Pei et al. Supplementary Figure S1
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Supplementary Data 1.
Supplementary Data 1. Evaluation of the effects of number of F2 progeny to be bulked (n) and average sequencing coverage (depth) of the genome (G) on the levels of false positive SNPs (SNP index = 1).
[Presented by: Andrew Howlett, Cruise Slater, Mahmud Hasan, Greg Dale]
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Mutational Dissection [Presented by: Andrew Howlett, Cruise Slater, Mahmud Hasan, Greg Dale] Introduction What is the point of Mutational Dissection? It allows understanding of normal biological functions
Supplementary Methods
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How to deal with the microarray results.
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