Protocol to evaluate a lineage marking system in the Drosophila testis

Accurate interpretation of genetic lineage tracing data is often confounded by the specificity and sensitivity of promoter activity and recombination rates. Thus, reported values may misrepresent targeted event frequency. Here, we present a protocol to validate a genetic tool for marking “dedifferentiation” in Drosophila testes. We describe steps for long-term live imaging to observe reporter activation and detail procedures to estimate false-positive and false-negative rates by integrating these datasets into a mathematical model. This approach enables rigorous evaluation of system performance. For complete details on the use and execution of this protocol, please refer to Bener et al. 1 • Strategies for designing genetic tools to detect dedifferentiated germline stem cells • Instructions for extended live imaging to monitor genetic marking • Estimating lineage tracing specificity and efficiency via mathematical modeling Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Accurate interpretation of genetic lineage tracing data is often confounded by the specificity and sensitivity of promoter activity and recombination rates. Thus, reported values may misrepresent targeted event frequency. Here, we present a protocol to validate a genetic tool for marking “dedifferentiation” in Drosophila testes. We describe steps for long-term live imaging to observe reporter activation and detail procedures to estimate false-positive and false-negative rates by integrating these datasets into a mathematical model. This approach enables rigorous evaluation of system performance.