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

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...

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Detalles Bibliográficos
Autores principales: Rondelet, Arnaud, Pozniakovsky, Andrei, Namboodiri, Devika, Cardoso da Silva, Richard, Singh, Divya, Leuschner, Marit, Poser, Ina, Ssykor, Andrea, Berlitz, Julian, Schmidt, Nadine, Röhder, Lea, Vader, Gerben, Hyman, Anthony A, Bird, Alexander W
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Life Science Alliance LLC 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756954/
https://www.ncbi.nlm.nih.gov/pubmed/33293335
http://dx.doi.org/10.26508/lsa.202000836
Descripción
Sumario: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.