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• W3113201870 abstract "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." @default.
• W3113201870 created "2020-12-21" @default.
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• W3113201870 date "2020-12-08" @default.
• W3113201870 modified "2023-09-23" @default.
• W3113201870 title "ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes" @default.
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• W3113201870 doi "https://doi.org/10.26508/lsa.202000836" @default.
• W3113201870 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7756954" @default.
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