TY - JOUR T1 - ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes JF - Life Science Alliance JO - Life Sci. Alliance DO - 10.26508/lsa.202000836 VL - 4 IS - 2 SP - e202000836 AU - Arnaud Rondelet AU - Andrei Pozniakovsky AU - Devika Namboodiri AU - Richard Cardoso da Silva AU - Divya Singh AU - Marit Leuschner AU - Ina Poser AU - Andrea Ssykor AU - Julian Berlitz AU - Nadine Schmidt AU - Lea Röhder AU - Gerben Vader AU - Anthony A Hyman AU - Alexander W Bird Y1 - 2021/02/01 UR - https://www.life-science-alliance.org/content/4/2/e202000836.abstract N2 - 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. ER -