TY - JOUR
T1 - ESI mutagenesis
T2 - A one-step method for introducing mutations into bacterial artificial chromosomes
AU - Rondelet, Arnaud
AU - Pozniakovsky, Andrei
AU - Namboodiri, Devika
AU - Silva, Richard Cardoso Da
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.
N1 - Funding Information: We thank Mihail Sarov for helpful comments on the manuscript. This work was supported by a Worldwide Cancer Research project grant 16-0093 to AW Bird. The Vader laboratory was financially supported by the European Research Council (ERC Starting Grant URDNA, agreement number 638197, to G Vader) and the Max Planck Society. Publisher Copyright: © 2021 Rockefeller University Press. All rights reserved. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/2
Y1 - 2021/2
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.
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.
UR - http://www.scopus.com/inward/record.url?scp=85097515050&partnerID=8YFLogxK
U2 - https://doi.org/10.26508/LSA.202000836
DO - https://doi.org/10.26508/LSA.202000836
M3 - Article
C2 - 33293335
SN - 2575-1077
VL - 4
JO - Life Science Alliance
JF - Life Science Alliance
IS - 2
M1 - e202000836
ER -