Genome-Scale Mutagenesis - PowerPoint PPT Presentation

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Genome-Scale Mutagenesis

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Introduction Model systems Yeast Mouse Implications for science Genotype - Phenotype Reverse Genetics - Forward Genetics Flow of genetic information Flow of genetic ... – PowerPoint PPT presentation

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Title: Genome-Scale Mutagenesis


1
Genome-Scale Mutagenesis
  • Introduction
  • Model systems
  • Yeast
  • Mouse
  • Implications for science

2
Genotype - Phenotype
  • what is a gene?
  • genes to function
  • how do you study this?

3
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
4
Flow of genetic information
Genotype
Phenotype
GeneDNA
RNA
Protein Function
1
1
1
5
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
6
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
7
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

8
Yeast mutagenesis
  • Random, insertional mutagenesis
  • No prior knowledge involved
  • Multiple mutant alleles possible
  • Targeted mutagenesis
  • Precise, null mutations

9
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

10
Transposon mutagenesis in yeast
11
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

12
Transpson-mediated mutations in yeast
13
Gene-specific mutations in yeast
14
Directed mutations in yeast
15
Classification of gene functions in yeast
16
Aneuploidy in yeast deletion strains
17
Segmental aneuploidy and mRNA expression
18
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

19
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

20
Gene-trap vector
21
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

22
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

23
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

24
Mouse ENU mutagenesis
25
F1 ENU mutants with visible phenotypes
(a) Nanomouse (b) dominant spotting (c)
microphthalmia mutant (d, e) Batface
26
F1 screening protocols
27
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28
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29
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

30
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

31
Summary
  • Efficient functional genomics approach?
  • No prior knowledge of phenotype
  • Genome-scale mutant resources
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