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Journal Article

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

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Pozniakovsky,  Andrei I.
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Poser,  Ina
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

/persons/resource/persons219687

Ssykor,  Andrea
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

/persons/resource/persons219253

Hyman,  Anthony
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

/persons/resource/persons219013

Bird,  Alexander W.
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Citation

Rondelet, A., Pozniakovsky, A. I., Namboodiri, D., Silva, R. C. d., Singh, D., Leuschner, M., et al. (2021). ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes. Life science alliance, 4(2): e202000836. doi:10.26508/lsa.202000836.


Cite as: https://hdl.handle.net/21.11116/0000-0008-DA9E-1
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.