ESI mutagenesis: A one-step method for introducing mutations into bacterial artificial chromosomes

Arnaud Rondelet, Andrei Pozniakovsky, Devika Namboodiri, Richard Cardoso Da Silva, Divya Singh, Marit Leuschner, Ina Poser, Andrea Ssykor, Julian Berlitz, Nadine Schmidt, Lea Röhder, Gerben Vader, Anthony A. Hyman, Alexander W. Bird*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although 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 exogenous/synthetic intronization (ESI) 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.

Original languageEnglish
Article numbere202000836
JournalLife Science Alliance
Volume4
Issue number2
DOIs
Publication statusPublished - Feb 2021

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