RT Journal Article
SR Electronic
T1 ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202000836
DO 10.26508/lsa.202000836
VO 4
IS 2
A1 Rondelet, Arnaud
A1 Pozniakovsky, Andrei
A1 Namboodiri, Devika
A1 Cardoso da Silva, Richard
A1 Singh, Divya
A1 Leuschner, Marit
A1 Poser, Ina
A1 Ssykor, Andrea
A1 Berlitz, Julian
A1 Schmidt, Nadine
A1 Röhder, Lea
A1 Vader, Gerben
A1 Hyman, Anthony A
A1 Bird, Alexander W
YR 2021
UL https://www.life-science-alliance.org/content/4/2/e202000836.abstract
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.