Cymbeline T. Culiat, Ph. D. Functional Genomics Group Life Sciences Division
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 1 Functional Annotation of Human Genome Sequence
• Assigning function to ~30,000 - 50,000 genes • Identifying regulatory elements (unknown number) • Experimentally validating predictions of gene function • Detailed analysis of mechanisms of function (complex biological pathways)
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 2 Mouse Mutagenesis: An Indispensable Approach
• Examine function by observing consequences of disrupting a gene in a mammalian system • The mouse remains the premier mammalian animal model • Mutant mouse serves as reagent for both basic and pharmaceutical studies • Often yields very surprising results!
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 3 GABRB3 in Craniofacial Development
• GABRB3, a neurotransmitter receptor molecule was initially predicted to be a gene playing a major role in mental retardation
• The major role of this gene in facial clefting was first demonstrated at ORNL using a series of mouse mutations and gene-rescue experiments (Culiat et al., 1995, Nature Genetics) .
• A recent publication (Scapoli et al., 2002, Human Genetics) has linked the GABRB3 gene to non-syndromic cleft lip and palate in man.
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 4 Generating Mouse Mutations with ENU • N-ethyl-N-nitrosourea (ENU), an alkylating agent
• Potent mutagen of mouse spermatogonial stem cells
• Induces primarily single base pair changes in DNA William B Russell et al. (1979) PNAS 76:5918-5911. ORNL • Different mutations in the same gene with a range of consequences- an allelic series
• Slightly altered proteins for protein- complex analysis
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 5 Large- scale ENU-mutagenesis Programs
• Publicly funded - USA (ORNL, Jackson Lab, Baylor Univ, NIH consortia) - United Kingdom (MRC, Harwell) - Germany - Japan - Canada - Australia • Privately funded - Novartis - GlaxoSmithKline - Pharmacia - Ingenium Pharmaceuticals AG - Hypnion Inc. - Celltech R&D
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 6 Temperature-gradient capillary electrophoresis (TGCE)
Q. Gao and E. S. Yeung High-throughput detection of unknown mutations by using multiplexed capillary electrophoresis with poly(vinylpyrrolidone) solution. Analytical Chemistry 2000, 72: 2499-2506 Ames National Laboratory, USDOE Dept of Chemistry, Iowa State University
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 7 Mutation Scanning using the SCE9610 (SpectruMedix LLC)
• High-throughput - twelve 96-well plates/day - 150-800 bp PCR fragments with different Tms • Highly sensitive - low sample requirement (~200 pg/µl) - 97% accuracy, 3% false positive rate • Simple and low-cost sample preparation - mouse tail DNA preps for PCR templates - unpurified PCR reactions for TGCE scanning - ethidium bromide laser-induced fluorescence • Multiplexing - different amplicons in single capillary - pooling of samples Li, Q., Liu, Z., Monroe, H. and Culiat, C. T. (2002) Electrophoresis 23:1499-1511
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 8 Mutation Scanning with TGCE
Heteroduplex formation Separation by TGCE
• Retarded mobility of heteroduplexes compared to homoduplexes • Optimal temperature for resolution is sequence-dependent
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 9 1 23 4 5 6 7 8 910 11 12 A B C 5961SB D Optimizing TGCE E F G H 6287SB Fah 5961SB ; 493 bp, exon 7, G to A Fah 6287SB ; 340 bp, exon 6, A to G
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 10 Regional Mutagenesis: Chromosome 7 p region
Ldh1 Saa3 Prmt3? Nell1
19q 11p15-p14 15q13-q11
Wildtype littermate l7R6 mutant
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OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 11 Craniosynostosis: Premature Fusion of Skull Bones
Pre-surgery Pre-surgery Post-surgery
• Intracranial pressure • Impaired cerebral flow • Impaired vision and hearing • Learning disabilities • Psychological defects Post-surgery
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 12 A Gene-Driven Approach of Mouse Mutagenesis
Gene Selection Mutation Scanning Rederivation Recover mutant A set of genes in any *CMMB:Cryopreserved mouse by IVF and/or biological pathway of Mutant Mouse Bank interest ICSI A bank of DNAs, sperm and tissues from mice mutagenized by ENU. • Skin Carcinogenesis • Craniofacial Birth Defects • Low-dose Radiation Susceptibility Functional Studies • Chemical and Pathogen Susceptibility • Intracellular Transport Genetics • Aging and DNA repair Pathology • Maternal Effects on DNA Repair Physiology Biochemistry *Initial Funding: LDRD PIs: E. Michaud, C. Culiat and E. Rinchik
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY Generating the CMMB Resource
ENU
PCR amplification
Genomic DNA Tail biopsies cDNA Scanning/TGCE & DNA Sequencing Pooled tissues for DNA/RNA/proteins 5000 male mice Skin, brain, heart, lungs, liver, intestine, spleen, kidneys, testes Reconstitute Spermatozoa Mutant Mice from Sperm
OAK RIDGE NATIONAL LABORATORY Cryopreservation U. S. DEPARTMENT OF ENERGY 14 First 2780 (55%) mice have been banked Established conditions for high-throughput PCR and TGCE mutation scanning A 25-Mb screen under way; largest gene- driven mutation scanning experiment attempted to date. Re-derivation from frozen sperm set-up FileMaker Pro Database
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 15 Regional Whole-genome Phenotype-driven Gene- and sequence- Chrs 7, 10, 15, and X driven Aberrant Phenotype Gene Gene Aberrant Phenotype
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X CMMB G4 TGCE is a breakthrough in finding ENU-induced mutations in the mouse genome!
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 16 ORNL Staff ORNL Technical Staff Ed Michaud K. T. Cain Sarah Shinpock Bem Culiat Debra Carpenter Ann Wymore Dabney Johnson Lori Easter Marilyn Kerley Gene Rinchik Alysyn Gardner Lori Hughes Mitch Klebig Kay Houser Shen Lu Jay Snoddy Bob Olszewski Melissa York Frank Larimer Irina Pinn Carmen Foster Beverly Stanford Ginger Shaw SpectruMedix LLC Qingbo Li Zhaowei Liu DOE-BER LDRD Program Heidi Monroe Marvin Frazier ZY Guo Kevin Gutshall
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 17 heterozygotes controls
200bp, G>A @ 34bp R4/6 R4
200bp, T>C @ 66bp R4/8 R8
200bp, G>A @ 34bp & R6/8 R6 T>C @ 66bp
493bp, G>A @ 268bp B3*/4* B3*
341bp, G>C @ 98bp C6* C5*
341bp, G>C @ 98bp C8*/9* C8*
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 18 A Crude Estimate of Mutation Frequency in the CMMB
• 1 mutation per 0.5 -2.5 Mb - whole animal mutagenesis (Russell, Rinchik, ORNL) - mutagenized ES cell bank (T. Magnuson, UNC) - whole animal mutagenesis (S. Brown, Harwell)
• An “average” gene (2.5 kb DNA) will have ~5 to 25 independent ENU-induced mutations
• Single SCE9610 can screen an amplicon/ week in entire bank
OAK RIDGE NATIONAL LABORATORY U. S. DEPARTMENT OF ENERGY 19