Efficient Gibson Assembly of Ultra-Large cDNAs: Generation of Full-length Wild-Type and Mutated futsch transgenes in Drosophila

Abstract

Research on ultra-large proteins (>5,000 amino acids) is often hindered by the lack of available full-length cDNA constructs and the technical challenges inherent with their cloning. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp), the homologue of mammalian MAP1B, using Gibson Assembly. These resulting cDNA were used to generate UAS-futsch transgenes. When expressed in neurons, the wild type transgenic Futsch associated with microtubule and rescued the synaptic morphological defects observed in futschK68 mutants. This approach substantially reduces the time and complexity compared with traditional cloning techniques. Furthermore, I highlight common pitfalls encountered during the cloning process and provide practical solutions to enhance cloning efficiency. This protocol serves as a valuable reference for researchers aiming to clone other ultra-large cDNAs for functional and structural studies.