Lecture 4. Drosophila transgenes and making mosaic flies. 18.10.24 The original Drosophila transformation method. (Rubin, Spradling, Gehring 1980s) • • • • GAL4-UAS binary expression system in flies • Yeast GAL4 gene encodes a positive regulatory protein that turns on transcription of GAL genes to grow on galactose • Mark Ptashne with graduate students Cynthia Wohlberger, Liam Keegan, Ed Giniger at Harvard, 1982 • • • Enhancer trapping. Remobilization of a P-element transgene having promoterless GAL4, using cross to Jumpstarter to supply transposase. Screening for wing imaginal disc expression patterns in new promoterless GAL4 insertion lines • Induction of ectopic eyes by GAL4-targeted expression of the eyeless gene in Drosophila • The image “file:///H:/Ptashne%20lab%20photos/eyefig1.GIF” cannot be displayed, because it contains errors. The image “file:///H:/Ptashne%20lab%20photos/eyefig2.GIF” cannot be displayed, because it contains errors. The image “file:///H:/Ptashne%20lab%20photos/eyefig3.GIF” cannot be displayed, because it contains errors. • Hormone- activated GAL4, GeneSwitch •G Tet-off • GAL4-driven tissue-targeted RNAi knockdown of gene expression • • Why we need to make genetically mosaic animals • •Probably missing many female sterile mutants here because homozygous mothers are not obtainable. D:\Harish\CB32-online figure\CB32-online figure\CH-01\CB32-WieschausFig02-revised.tif Making mosaics with clones of mutant cells in Drosophila •Many female sterile mutants could have been missed because homozygous mutant mothers cannot be obtained; the homozygous mutant mothers die as embryos or larvae. • •Such mutants are zygotic lethals. For zygotic lethals in general, studying their effects in detail requires making mosaic animals having clones of homozygous mutant tissue in heterozygous eggs, embryos, larvae or adult flies (somatic clones). • •If the lethal mutant has a female sterile defect because the mutation removes something required in nurse cells or oocytes, then it has to be studied using clones made in the germline (germline clones) Mitotic recombination Somatic clones. Twin spots on fly cuticle show recessive mutant markers in side-by side clones arising from a single mitotic recombination event (Curt Stern, 1930). Germline clones in ovaries using the dominant female sterile (DFS) mutant OvoD • MR = mitotic recombination to form clones of recombinant cells • • • • • Making somatic clones with Flp/FRT • Examples of somatic FRT clones and screens • • • Mosaic Analysis with a Repressible Marker (MARCM) combines Flp/FRT with GAL4/UAS • GAL80 removal by Flp away • • https://www.researchgate.net/profile/Radoslaw-Ejsmont/publication/262339705/figure/fig1/AS:60169894 2869519@1520467615399/Mosaic-analysis-with-a-repressible-cell-marker-MARCM-A-Gal4-transcription-fac tor.png Making germline clones efficiently with Flp/FRT •Probably missing many female sterile mutants here because homozygous mothers are not obtainable. D:\Harish\CB32-online figure\CB32-online figure\CH-01\CB32-WieschausFig02-revised.tif Mitotic recombination Somatic clones. Twin spots on fly cuticle show recessive mutant markers in side-by side clones arising from a single mitotic recombination event (Curt Stern, 1930). Germline clones in ovaries using the dominant female sterile (DFS) mutant OvoD • Perrimon came for Madeleine Gans lab in Paris. Screen for female sterile mutants on the X chromosome gave OvoD mutant. Mutagenized X chromosomes were crossed to Compound X, a balancer with fused copies of the X, used to keep stocks of female-lethal or female-sterile mutants. Female sterile mutants on the X chromosome included a dominant female sterile that is now called OvoD • • • • Flp/FRT clones are used to study stem cell maintenance in germline stem cells and in somatic stem cells • Mitotic recombination Somatic clones. Twin spots on fly cuticle show recessive mutant markers in side-by side clones arising from a single mitotic recombination event (Curt Stern, 1930). Germline clones in ovaries using the dominant female sterile (DFS) mutant OvoD • • • • Reading •The art and design of genetic screens: Drosophila melanogaster •D St Johnston - Nature reviews genetics, 2002 - nature.com • •[PDF] Creating mosaics in Drosophila •N Perrimon - International Journal of Developmental …, 1998 - genepath.med.harvard.edu • •[PDF] GAL4 system in drosophila: A fly geneticist's swiss army knife •JB Duffy - genesis, 2002 - fenix.ciencias.ulisboa.pt • • • • • • • • • Uses of GAL4 Number 1: The yeast two hybrid system for identification of interacting proteins. • Can be used on a genomic scale to test all proteins against all others. Target protein fused to DNA-binding domain in cells of one mating type mated to a library of activating region fusion proteins. Uses of GAL4: GAL4 activates transcription from UASG in Drosophila. Widely used for targeted protein expression in Drosophila (GAL4-UAS two-component system). • • • • • Flp-out, GAL4-on • UAS Flp-on • GAL80 removal by Flp away • • https://www.researchgate.net/profile/Radoslaw-Ejsmont/publication/262339705/figure/fig1/AS:60169894 2869519@1520467615399/Mosaic-analysis-with-a-repressible-cell-marker-MARCM-A-Gal4-transcription-fac tor.png Ey-Flp, directed mosaics • Modifier screen in eyes •