Genome-Scale Mutagenesis

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Genome-Scale Mutagenesis

• Introduction
• Model systems
• Yeast
• Mouse
• Implications for science

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Genotype - Phenotype

• what is a gene?
• genes to function
• how do you study this?

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Reverse Genetics - Forward Genetics
Reverse
Phenotype Inherited disease Sickle cell
anemia Cystic fibrosis Retinoblastoma
Breast Cancer
Genotype Single gene locus Hemoglobin CFTR
Rb BRCA1, 2
Forward
Genotype
Phenotype
mutagenesis
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Flow of genetic information
Genotype
Phenotype
GeneDNA
RNA
Protein Function
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Flow of genetic information
Genotype
Phenotype
GeneDNA
RNA
Protein Function
Tissue-specific expression Inducible
expression Alternative splicing
Mutation/ Polymorphism
Post-translation modification Protein-protein
interaction
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Flow of genetic information
Genotype
Phenotype
GeneDNA
RNA
Protein Function
Tissue-specific expression Inducible
expression Alternative splicing
Mutation/ Polymorphism
Post-translation modification Protein-protein
interaction
Human Genome Project
SNP Detection
cDNA Microarrays
Proteomics Two-hybrid
Mutant Phenotype
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Models for Genetic Analyses

• E.coli 3600 genes
• Yeast 6400
• C.elegans 13,500
• Drosophila 14,000 - 180 Mbps
• Zebrafish 25,000?
• Mouse 30-40K? - 3000 Mbps
• Human 30-40K? - 3000 Mbps

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Yeast mutagenesis

• Random, insertional mutagenesis
• No prior knowledge involved
• Multiple mutant alleles possible
• Targeted mutagenesis
• Precise, null mutations

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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

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Transposon mutagenesis in yeast
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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

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Transpson-mediated mutations in yeast
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Gene-specific mutations in yeast
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Directed mutations in yeast
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Classification of gene functions in yeast
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Aneuploidy in yeast deletion strains
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Segmental aneuploidy and mRNA expression
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Mouse mutants

• Natural, spontaneous mutants
• Null mutation by gene-knockout in ES cells
• 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

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Gene-Trap in ES cells

• Random, insertional mutagenesis using a DNA
  fragment having a reporter or selectable marker
• Marker is inserted into gene gt null mutation
• Fusion transcript between gene and marker
• Low mutation frequency
• Lexicon Genetics, 10,000 ES clones

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Gene-trap vector
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Mouse ENU mutagenesis

• N-ethyl-N-nitrosourea (ENU)
• Very high mutation rate
• ENU generates point mutations
• 44 A/T gt T/A
• 38 A/T gt G/C
• Many types of mutations possible, as well as null
• Loss-of-function, gain-of-function

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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

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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

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Mouse ENU mutagenesis
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F1 ENU mutants with visible phenotypes
(a) Nanomouse (b) dominant spotting (c)
microphthalmia mutant (d, e) Batface
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F1 screening protocols
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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

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Models for Genetic Analyses

• E.coli 3600 genes
• Yeast 6400
• C.elegans 13,500
• Drosophila 14,000 - 180 Mbps
• Zebrafish 25,000?
• Mouse 30-40K? - 3000 Mbps
• Human 30-40K? - 3000 Mbps

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Summary

• Efficient functional genomics approach?
• No prior knowledge of phenotype
• Genome-scale mutant resources