Singh, Samarjeet; Giesecke, Astrid ; Damulewicz, Milena; Fexova, Silvie; Mazzotta, Gabriella M.; Stanewsky, Ralf ; Dolezel, David: New "Drosophila" Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of "Timeless"
Inhalt
- Materials and Methods
- Fly Strains and per, cry, tim Combinations
- Locomotor Activity Measurement and Analysis
- Interspecific Comparison of TIM Proteins
- Nuclear Export Signal (NES) Prediction
- Prediction of Phosphorylation Sites
- Gene Editing Inducing Non-homologous-End-Joining (NHEJ)—gRNA Design
- Gene Editing Inducing Homology Directed Repair (HDR)—gRNA Design
- Gene Editing Inducing HDR—Donor Plasmid Construction
- Transgenesis for NHEJ Mutagenesis
- Transgenesis for HDR Experiments
- Genetic Crosses Inducing NHEJ
- Behavioral Screening in NHEJ Experiments
- Molecular Screening in HDR Experiments
- Sequencing of Mutated Region
- Immunocytochemistry
- Quantitative Comparison of Immunofluorescence Values
- Compensation for Different Staining Affinity After Antibody Re-use
- Statistical Analysis
- Results
- Genetic Interaction Between tim, cry, and per
- timblind Is a Temperature Compensation Mutant
- Step-Up Protocol for Detection of Temperature Compensation Mutants
- CRISPR/CAS9 Targeted Mutagenesis of tim
- Functional Characterization of Novel Mutant Lines
- Discussion
- Targeted Screen for Temperature Compensation Mutants
- Timeless in Temperature Compensation of the Circadian Clock
- Data Availability Statement
- Author Contributions
- Funding
- Acknowledgments
- Supplementary Material
- References