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 Phenotype Inherited disease Sickle cell anemia Cystic fibrosis Retinoblastoma Breast Cancer Genotype Single gene locus Hemoglobin CFTR Rb BRCA1, 2 GenotypePhenotype mutagenesis Reverse: Forward:

4. Flow of genetic information Gene:DNARNAProtein: Function GenotypePhenotype 111

5. Flow of genetic information Mutation/ Polymorphism Tissue-specific expression Inducible expression Alternative splicing Post-translation modification Protein-protein interaction GenotypePhenotype Gene:DNARNAProtein: Function

6. Flow of genetic information Mutation/ Polymorphism Tissue-specific expression Inducible expression Alternative splicing Post-translation modification Protein-protein interaction GenotypePhenotype Gene:DNARNAProtein: Function Human Genome Project SNP Detection cDNA Microarrays Proteomics Two-hybrid Mutant Phenotype

7. Models for Genetic Analyses E.coli3600 genes Yeast6400 C.elegans13,500 Drosophila14,000 - 180 Mbps Zebrafish25,000? Mouse30-40K? - 3000 Mbps Human30-40K? - 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. 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 > 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 > T/A –38% A/T > 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

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.coli3600 genes Yeast6400 C.elegans13,500 Drosophila14,000 - 180 Mbps Zebrafish25,000? Mouse30-40K? - 3000 Mbps Human30-40K? - 3000 Mbps

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