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Genome-Scale Mutagenesis

Introduction Model systems Yeast Mouse Implications for science. Genome-Scale Mutagenesis. what is a gene? genes to function how do you study this?. Genotype - Phenotype. Reverse Genetics - Forward Genetics. Reverse:. Genotype Single gene locus Hemoglobin CFTR Rb

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Genome-Scale Mutagenesis

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

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

  3. 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 Forward: Genotype Phenotype mutagenesis

  4. Flow of genetic information Genotype Phenotype Gene:DNA RNA Protein: Function 1 1 1

  5. Flow of genetic information Genotype Phenotype Gene:DNA 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 Gene:DNA 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. 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 Models for Genetic Analyses

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

  9. 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

  10. Transposon mutagenesis in yeast

  11. 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 Transposon mutagenesis in yeast

  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. 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 Mouse mutants

  19. 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 in ES cells

  20. Gene-trap vector

  21. 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 Mouse ENU mutagenesis

  22. 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 Allelic Series - qk

  23. 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 Full genome mutagenesis using ENU

  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. 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 Mapping heterozygous ENU mutations

  28. 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 Models for Genetic Analyses

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

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