Researchers from New York University have developed a new protocol to generate gene-specific split-GAL4 lines in Drosophila melanogaster (fruit fly) using genetic crosses, which offers a cost-effective alternative to traditional microinjection methods. The team, led by Dr. Yu-Chieh David Chen, including Siqi April Li, Hongzhou Gustave Li, Nathalie Shoji, and Dr. Claude Desplan, detailed their innovative approach in the journal STAR Protocols.
Dr. Chen stated, “This protocol significantly reduces the cost and complexity of creating gene-specific split-GAL4 lines, making it accessible for fly researchers focusing on various tissues.” The work is published in the peer-reviewed journal, STAR Protocols.
The researchers outlined a step-by-step method that includes four rounds of in vivo genetic crosses, PCR genotyping, and fluorescence imaging to ensure the correct orientation of split-GAL4 integration before establishing stable fly stocks. This process circumvents the need for microinjection, making it more accessible and less expensive.
One of the key findings of the study is that this new method is adaptable and can be used for generating a large batch of split-GAL4 hemi-drivers, which is essential for researchers working on different tissues in Drosophila. The researchers demonstrated the efficacy of their protocol by successfully generating split-GAL4 lines from existing MiMIC/CRIMIC lines, which are widely used in genetic studies of the fruit fly.
Professor Desplan emphasized the importance of this development: “Our approach offers a significant advantage over traditional methods, reducing the time and resources required to create gene-specific drivers. This will accelerate research in various fields, including neurobiology, developmental biology, and genetics.”
In their study, the researchers also highlighted the importance of phenotypic markers for tracking transgenes during genetic crosses. They noted that incorrect markers could indicate stock contamination, which could compromise the results. The protocol includes detailed instructions for maintaining fly stocks and preparing necessary buffers and reagents.
Moreover, the whole protocol can be employed as part of the Course-Based Undergraduate Research Experiences (CURE), which offers hands-on experience doing original research in fly genetics. The lines generated from the protocol will be broadly useful for generating cell-type-specific split-GAL4 lines to benefit researchers in the fly community.
In conclusion, the new protocol developed by Dr. Chen and his colleagues represents a significant advancement in the field of Drosophila genetics. By offering a cost-effective and efficient alternative to traditional methods, this protocol is set to enhance the capabilities of researchers worldwide, enabling more extensive and precise genetic studies.
Journal Reference
Li, S. A., Li, H. G., Shoji, N., Desplan, C., & Chen, Y. D. “Protocol for replacing coding intronic MiMIC and CRIMIC lines with T2A-split-GAL4 lines in Drosophila using genetic crosses.” STAR Protocols, 2023. DOI: https://doi.org/10.1016/j.xpro.2023.102706
About The Authors
Yu-Chieh David Chen, is an NIH F32 and K99/R00 fellow in Dr. Claude Desplan‘s lab at New York University. David was a Howard Hughes Medical Institute (HHMI) international student research fellow and did his graduate study in Dr. Anupama Dahanukar’s lab at the University of California Riverside. His long-term research interest is to understand developmental processes and functional roles of primary senses: vision, touch, hearing, and chemosensation (taste and smell), which are essential for organisms to perceive and interact with the world. David is currently on the faculty job market, looking to find a place to launch his independent research program. His future research program lies in understanding molecular regulators underlying various developmental events, such as cell specification, axonal targeting, and synaptic partner matching, to form the functional sensory circuits in Drosophila. David is also passionate about teaching and mentoring the next generation of scientists. See his website for more information: ycdavidchen.com
Siqi(April) Li, got her biology degree (BA) at the College of Arts and Sciences at New York University. She is particularly passionate about molecular biology, as she seeks to understand how microscopic physical processes govern the behavior of biological systems at every possible scale. In the past, Siqi interned at a pharmaceutical company, where she was tasked with building an online platform that educates the general public about the discoveries found in academic literature. More recently, Siqi has worked at a neuropsychology lab as a research volunteer, assisting with a wide range of experimental procedures. She joined Desplan’s lab in Fall 2022, working with David Chen to generate split-GAL4 genetic tools to label novel cell types in the developing Drosophila visual system. Inspired by the people she’s met along this journey, Siqi is pursuing her PhD at Icahn School of Medicine at Mount Sinai starting in Fall 2024.
Hongzhou (Gustave) Li obtained a B.S. degree in neural science with honors at New York University Shanghai in 2024. He gained interest in neurobiology from the intricate biological pathways that contribute to brain development and function, and he is passionate about understanding the brain from a bottom-up perspective. During his undergraduate study, Gustave worked in Dr. Claude Desplan’s lab at NYU Biology and was directly mentored by postdoctoral researcher Dr. Yu-Chieh David Chen, where he conducted research on generating genetic tools labeling individual neuronal cell types in the Drosophila central nervous system. He presented his work at multiple academic conferences and was awarded the Best Poster Presentation in Developmental Biology and Genetics at the 2023 Annual Biomedical Research Conference for Minoritized Scientists (ABRCMS). Starting in the Fall of 2024, he will pursue a PhD in neurobiology at the California Institute of Technology. He plans to enter academia and become an independent researcher after PhD.
