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ESI mutagenesis: A one-step method for introducing point mutations into bacterial artificial chromosome transgenes

By Arnaud Rondelet, Andrei Pozniakovsky, Marit Leuschner, Ina Poser, Andrea Ssykor, Julian Berlitz, Nadine Schmidt, Anthony Hyman, Alexander W. Bird

Posted 16 Nov 2019
bioRxiv DOI: 10.1101/844282

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. While homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call ESI (Exogenous/Synthetic Intronization) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI-mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

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