ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.
Journal
Life science alliance
ISSN: 2575-1077
Titre abrégé: Life Sci Alliance
Pays: United States
ID NLM: 101728869
Informations de publication
Date de publication:
02 2021
02 2021
Historique:
received:
01
07
2020
revised:
23
11
2020
accepted:
23
11
2020
entrez:
9
12
2020
pubmed:
10
12
2020
medline:
14
9
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 33293335
pii: 4/2/e202000836
doi: 10.26508/lsa.202000836
pmc: PMC7756954
pii:
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : European Research Council
ID : 638197
Pays : International
Informations de copyright
© 2020 Rondelet et al.
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