PT  - JOURNAL ARTICLE
AU  - Rondelet, Arnaud
AU  - Pozniakovsky, Andrei
AU  - Namboodiri, Devika
AU  - Cardoso da Silva, Richard
AU  - Singh, Divya
AU  - Leuschner, Marit
AU  - Poser, Ina
AU  - Ssykor, Andrea
AU  - Berlitz, Julian
AU  - Schmidt, Nadine
AU  - Röhder, Lea
AU  - Vader, Gerben
AU  - Hyman, Anthony A
AU  - Bird, Alexander W
TI  - ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
AID  - 10.26508/lsa.202000836
DP  - 2021 Feb 01
TA  - Life Science Alliance
PG  - e202000836
VI  - 4
IP  - 2
4099  - http://www.life-science-alliance.org/content/4/2/e202000836.short
4100  - http://www.life-science-alliance.org/content/4/2/e202000836.full
SO  - Life Sci. Alliance2021 Feb 01; 4
AB  - 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.