The protozoan parasite Leishmania mexicana serves as a widely used model for studying trypanosomatid biology, yet the demand for stable, high-intensity fluorescent tools for precise subcellular protein localization remains unmet. In this study, we developed a versatile molecular toolbox by re-engineering the pLEXSY-hyg2.1 vector to express mNeonGreen (mNG), a next-generation fluorophore with superior brightness and photostability. Using a modular cloning strategy, we introduced a customized multiple cloning site (MCS) upstream of the mNG sequence to facilitate seamless N-terminal tagging of target proteins. The construct was integrated into the 18S rRNA locus via homologous recombination to ensure stable, constitutive expression. As a proof-of-concept, we fused a flagellar marker to the mNG reporter, resulting in a transgenic line exhibiting robust and specific subcellular fluorescence without compromising cellular fitness. Our results demonstrate that this integration-based system provides a highly efficient and stable platform for visualizing protein distribution within Leishmania. This tool significantly simplifies the generation of reporter strains and will facilitate high-resolution imaging studies of parasite organelle dynamics and functional genomics.
Lei et al. (Tue,) studied this question.