1. Announcements Exam I next Tuesday (2/17) Will cover all material through lecture this week

2. CONDITIONAL MUTATIONS & MOSAIC ANALYSIS

3. CONDITIONAL MUTATIONS Permit temporal and/or tissue specific control over the loss of gene activity

4. Purposes of Conditional Mutations Examine later and/or tissue-specific functions of a gene required for viability Bypass lethality to examine later function Determine where gene function is required Find which tissue is the source of gene activity Determine “autonomy” of gene function

5. Conditional Alleles Small protein changes –Heat sensitive –Cold sensitive Engineered domains –Drug-dependent Recombination-induced genome modification (Mosaic analysis)

6. Isolation of conditional alleles Typically, generated at random –Point mutants –Labor intensive to identify –Rare, so not often done in multicellular organisms Can be created from known conditional alleles in similar genes

7. Directed creation of ts alleles

8. MOSAIC ANALYSIS

9. MOSAIC ANALYSIS - WHY? Purposes of Mosaic Analysis Examine later and/or tissue-specific functions of a gene required for viability Bypass lethality to examine later function Determine where gene function is required Find which tissue is the source of gene activity Determine “autonomy” of gene function Cell lineage analysis

10. Recombination-Induced Genome Modification Mediated by mitotic recombination Interchromosomal Intrachromosomal Must be induced (not normal) Site-specific enzymes »FLP recombinase »Cre recombinase

12. Mouse Mosaics: Cre/lox Recombination

13. There are various designs of targeting vector that can be used to generate loss- or gain-of- function alleles in ES cells. ©2008 by American Physiological Society Adams D J, van der Weyden L Physiol. Genomics 2008;34:225-238 LOX FRT

17. Ligand-dependent Cre Recombinase

18. Tamoxifen-dependent Cre

19. Drosophila mosaics

21. Drosophila- FLP/FRT-Mediated Mitotic Recombination (Interchromosomal)

23. SEM of Drosophila Compound Eye

24. w+w+ w-w- FRT FLP FLP/FRT Targeted Mitotic Recombination Recombination Step

25. w+w+ w-w- FRT w + / w - red eyes w + / w - red eyes FLP/FRT Targeted Mitotic Recombination Segregation Option I: Outsides vs. Insides

26. w+w+ w-w- FRT w + / w + red eyes w - / w - white eyes FLP/FRT Targeted Mitotic Recombination Segregation Option II: Tops vs. Bottoms

28. What do we need? Mutation of interest distal to the FRT FRT near the centromere (preferably) Source of FLP recombinase Cell autonomous marker of genotype

29. Employing cell markers for mitotic recombination

31. Eye Differentiation in Drosophila

32. RTK Signal Transduction Pathway

35. MEK + MEK - FRT FLP FLP/FRT Targeted Mitotic Recombination Recombination Step his-GFP

36. MEK + MEK - FRT MEK + /MEK - green cells MEK + /MEK - green cells FLP/FRT Targeted Mitotic Recombination Segregation Option I: Outsides vs. Insides his-GFP

37. MEK + MEK - FRT MEK + /MEK + green cells MEK - /MEK - white cells FLP/FRT Targeted Mitotic Recombination Segregation Option II: Tops vs. Bottoms his-GFP