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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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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
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author 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
author_facet 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
author_sort Rondelet, Arnaud
collection PubMed
description 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.
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spelling pubmed-77569542020-12-30 ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes 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 Life Sci Alliance Research Articles 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. Life Science Alliance LLC 2020-12-07 /pmc/articles/PMC7756954/ /pubmed/33293335 http://dx.doi.org/10.26508/lsa.202000836 Text en © 2020 Rondelet et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Articles
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
ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
title ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
title_full ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
title_fullStr ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
title_full_unstemmed ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
title_short ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
title_sort esi mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes
topic Research Articles
url 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
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