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

PhenotypeInherited disease Sickle cell anemia Cystic fibrosis Retinoblastoma Breast Cancer

GenotypeSingle gene locus Hemoglobin CFTR Rb BRCA1, 2

Genotype Phenotypemutagenesis

Reverse:

Forward:

Flow of genetic information

Gene:DNA RNA Protein: Function

Genotype Phenotype

1 1 1

Flow of genetic information

Mutation/Polymorphism

Tissue-specific expressionInducible expressionAlternative splicing

Post-translation modificationProtein-protein interaction

Genotype Phenotype

Gene:DNA RNA Protein: Function

Flow of genetic information

Mutation/Polymorphism

Tissue-specific expressionInducible expressionAlternative splicing

Post-translation modificationProtein-protein interaction

Genotype Phenotype

Gene:DNA RNA Protein: Function

HumanGenomeProject

SNPDetection

cDNAMicroarrays

ProteomicsTwo-hybrid

MutantPhenotype

Models for Genetic Analyses

• E.coli 3600 genes

• Yeast 6400

• C.elegans 13,500

• Drosophila14,000 - 180 Mbps

• Zebrafish 25,000?

• Mouse 30-40K? - 3000 Mbps

• Human 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– 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

• E.coli 3600 genes

• Yeast 6400

• C.elegans 13,500

• Drosophila14,000 - 180 Mbps

• Zebrafish 25,000?

• Mouse 30-40K? - 3000 Mbps

• Human 30-40K? - 3000 Mbps

Summary

• Efficient functional genomics approach?

• No prior knowledge of phenotype

• Genome-scale mutant resources