A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• Protein localization is crucial for understanding protein function.
• Current protein localization methods lag behind high-throughput genomics, creating bottlenecks.
• Tagging proteins in trypanosomatids is essential for post-genomic analysis.

Purpose of the Study:

• To develop efficient tools for protein tagging in trypanosomatids.
• To overcome limitations of existing methods, particularly in organisms with less efficient homologous recombination.
• To enable scalable, whole-genome protein tagging for functional studies.

Main Methods:

• Developed a plasmid system for long primer PCR tagging at endogenous loci.
• Created fusion PCR and specialized plasmids for gene tagging in Leishmania mexicana.
• Optimized primer design, PCR conditions, and workflow for reliability and scalability.

Main Results:

• Successfully generated transgenes encoding tagged proteins at endogenous loci.
• Developed a fusion PCR approach enabling gene tagging in Leishmania mexicana.
• Demonstrated a reliable, efficient, and scalable system for whole-genome tagging in trypanosomatids.

Conclusions:

• The developed tools overcome previous limitations in trypanosomatid protein tagging.
• This system facilitates efficient functional domain analysis and protein localization studies.
• Whole-genome protein tagging is now an achievable goal for advancing trypanosomatid research.