Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes

Analysis of gene function • Loss-of-function • Many gene knock-out have no obvious phenotypes • Redundancy? • No perfect redundancy => subtle phenotype • Gain-of-function • Many give observable phenotypes (e.g. oncogenes) • mis-expression • enhanced expression • ectopic expression • constitutive activation

Using Drosophila to analyze human gene function • Homologous gene in fly (FlyBase) • find fly mutation • study fly mutation • identify interacting genes • check in human for involvement of the interacting genes • Human gene family usually have more genes than in fly • easier to study in fly • can not address functional diversity

Clonal (mosaic) analysis • Analysis of gene with pleiotropic functions (e.g. at later stages) • Determine the site of gene function +/- +/+ -/-

Making a mitotic clone Random location FRT + marker FRT m hs-FLP HS FRT + marker + marker FRT + marker + marker m FRT m FRT m

loxP loxP Enhancer-Cre recombinase => tissue-specific knock-out

No fly homolog • Ectopic expression in fly => phenotype • Determine what process/pathway is affected • Similar function in human? • Search for interacting genes in fly • Similar gene interaction in human?

Clonal induction of expression X UAS Actin promoter CD2 STOP GAL4 GFP FRT FRT UAS hs-FLP random location; can sample all cells Enhancer-FLP

Targeted expression screen UAS Drives expression enhancers Endogenous gene UAS Drives anti-sense expression Cross with GAL4 lines => targeted expression

Flowchart for ectopic expression in fly • 1. Make UAS-X construct • 2. Microinjection to fly embryo • 3. Isolate transformants and set up balanced lines • 4. Drive expression with GAL4 lines • GMR-GAL4 • dpp-GAL4 • Eq-GAL4 5. Examine phenotype • what cell types are affected? • what cellular processes are affected? • fate transformation? • cell death or proliferation?

Using fly to study human gene function Fly homolog Fly mutant No fly homolog Misexpression in fly Phenotype in fly Function Interacting genes

利用果蠅研究人類疾病 人類MJD基因表現於果蠅造成類似的神經退化壞死現象正常的MJD基因致病的MJD基因

Coexpression of eyg and ey show Synergistic effect in eye induction dpp>eyg dpp>ey dpp>eyg+ey

Dorsal midline fusion of the thorax wt gv (eyg) Eq>hth

6. Predict interacting genes • based on knowledge in fly study • obtain relevant mutants • test for genetic interactions Modifier mutation (suppressor, enhancer) Second site mutation that modifies the mutant phenotype of the first gene a-/a+b-/b+ => mutant phenotype a-/a+b-/b- => stronger phenotype (dominant enhancer) a-/a-b-/b- => synergistic (more than additive) effect

Effect of X (ectopic expression) may be suppressed by l-o-f mutation in C A X B C D

7. Screen for modifier mutations • Screen for (EMS) mutations that enhance or suppress the ectopic expression phenotype • Map mutations by recombination or deficiency • Predict candidate genes from map location • Get mutants for candidate genes • Confirm genetic interactions • Similar relationship in human?

Reverse genetics: From a gene to function • 1. Candidate genes by location • Map position • FISH • mapped genomic clones • genome sequence • Known mutations mapped at same location • Molecular defect of the gene in mutants • Southern • expression

2. Gene knock-out by homologous recombination • 3. Insertional mutaenesis • screen by PCR • 4. Post-transcriptional inactivation • Antisense RNA • Ribozyme • RNA interference (RNAi, dsRNA)

5. Dominant-negative protein e.g. Transcription factor Domains: DNA-binding, Activation, Oligomerization D- A+ O+ oligomer without DNA-binding D+ A- O+ seq-specific repressor D+ A- O- blocks target sites

Analysis of gene function Loss-of-function Many gene knock-out have no obvious phenotypes