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Genome-Scale Mutagenesis
• Introduction
• Model systems
– Yeast
– Mouse
• Implications for science
Genotype - Phenotype
• what is a gene?
• genes to function
• how do you study this?
Reverse Genetics - Forward Genetics
Reverse:
Genotype
Single gene locus
Hemoglobin
CFTR
Rb
BRCA1, 2
Phenotype
Inherited disease
Sickle cell anemia
Cystic fibrosis
Retinoblastoma
Breast Cancer
Genotype
Phenotype
Forward:
mutagenesis
Flow of genetic information
Genotype
Phenotype
Gene:DNA
RNA
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Protein: Function
1
Flow of genetic information
Genotype
Gene:DNA
Mutation/
Polymorphism
Phenotype
RNA
Tissue-specific expression
Inducible expression
Alternative splicing
Protein: Function
Post-translation modification
Protein-protein interaction
Flow of genetic information
Genotype
Gene:DNA
Mutation/
Polymorphism
Human
Genome
Project
SNP
Detection
Phenotype
RNA
Tissue-specific expression
Inducible expression
Alternative splicing
cDNA
Microarrays
Protein: Function
Post-translation modification
Protein-protein interaction
Proteomics
Two-hybrid
Mutant
Phenotype
Models for Genetic Analyses
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E.coli
Yeast
C.elegans
Drosophila
Zebrafish
Mouse
Human
3600 genes
6400
13,500
14,000 - 180 Mbps
25,000?
30-40K? - 3000 Mbps
30-40K? - 3000 Mbps
Yeast mutagenesis
• Random, insertional mutagenesis
– No prior knowledge involved
– Multiple mutant alleles possible
• Targeted mutagenesis
– Precise, null mutations
Transposon mutagenesis in yeast
• In yeast, Ty1 transposon have been used
– Tends to insert into promoter regions
• Alternative: E.coli mTn3
– Mutagenize yeast genomic clones in E.coli
– Shuttle mutated DNA into yeast
Transposon mutagenesis in yeast
Transposon mutagenesis in yeast
• 92,500 plasmid preps of mutagenized yeast DNA
• Transformation resulted in growth of 11,232
haploid yeast strains
• Precise insertion site determined for 6,358 strains
• Insertion into 1917 ORFs
Transpson-mediated mutations in yeast
Gene-specific mutations in yeast
Directed mutations in yeast
Classification of gene functions in yeast
Aneuploidy in yeast deletion strains
Segmental aneuploidy and mRNA expression
Mouse mutants
• Natural, spontaneous mutants
• Null mutation by gene-knockout in ES cells
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Obtain genomic clones
Create targeting vector
Transfect and isolate ES mutant clone
Generate mice from ES clone
~2000 gene knockout mice lines
• Gene-trap in ES cells
Gene-Trap in ES cells
• Random, insertional mutagenesis using a DNA
fragment having a reporter or selectable marker
• Marker is inserted into gene > null mutation
• Fusion transcript between gene and marker
• Low mutation frequency
• Lexicon Genetics, 10,000 ES clones
Gene-trap vector
Mouse ENU mutagenesis
• N-ethyl-N-nitrosourea (ENU)
• Very high mutation rate
• ENU generates point mutations
– 44% A/T > T/A
– 38% A/T > G/C
• Many types of mutations possible, as well as null
– Loss-of-function, gain-of-function
Allelic Series - qk
• Quaking (qk) locus
• Homozygous qk-v (1Mb deletion)
– seizures and quaking, sterile males
• ENU alleles
– 4 are embryonic lethal
– 2 of 4, seizures or quaking in heterozygotes
– 1 allele, qk-e5, is viable
• extreme quaking and seizures, fertile males
Full genome mutagenesis using ENU
• ENU is a highly, efficient mutagen
– Especially on sperm, also ES cells
• Treatment of one animal generates 100 mutations
• Screen 300-500 mouse lines to test for new
mutations in every gene
• Mapping the mutation is the most difficult aspect
Mouse ENU mutagenesis
F1 ENU mutants with visible phenotypes
(a) Nanomouse
(b) dominant spotting
(c) microphthalmia mutant
(d, e) Batface
F1 screening protocols
Mapping heterozygous ENU mutations
• perform genetic mapping
– Need ~24 animals
– 8000 PCR reactions using known polymorphisms
– Mapping within 20 cM (20 Mbp)
• SNP mapping
• Expression profiling using microarrays
• Complementation by genomic, BAC clones
Models for Genetic Analyses
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•
E.coli
Yeast
C.elegans
Drosophila
Zebrafish
Mouse
Human
3600 genes
6400
13,500
14,000 - 180 Mbps
25,000?
30-40K? - 3000 Mbps
30-40K? - 3000 Mbps
Summary
• Efficient functional genomics approach?
• No prior knowledge of phenotype
• Genome-scale mutant resources