Generation of full-length wild-type and mutated futsch transgenes in Drosophila—efficient Gibson Assembly of ultra-large cDNAs

Document Type

Article

Publication Date

9-4-2026

Publication Title

Fly

Abstract

Drosophila Futsch is a key microtubule-associated protein (fly homolog of MAP1B) that regulates microtubule organization, synaptic terminal growth, and neuronal development. Functional analysis of futsch has long been limited by the inability to clone and express a full-length futsch transgene, owing to its exceptional size (~16.5 kb) and extensive repetitive sequences. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp) using Gibson Assembly. These resulting cDNAs 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 offers a broadly applicable and cost-effective framework for cloning otherwise intractable large cDNAs from Drosophila and other organisms.

PubMed ID

42697839

Volume

20

Issue

1

Publisher

Informa UK Limited, trading as Taylor & Francis Group

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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