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.
Highlights
The ability to precisely query functional hypotheses of protein function in cells requires the capacity to express rationally mutated proteins from genes under their native physiological regulation
We showed above that exogenous/synthetic intronization (ESI)-mutated Bacterial artificial chromosome (BAC) yield proteins of the expected size and the correct cellular localization, and that mutation may efficiently confer RNA-resistance or loss of interaction partner when analysed at the cell-population level
We have shown that by co-integrating a selectable marker located within an artificial intron, site-directed mutations can be quickly and efficiently introduced into BAC transgenes using a one-step recombineering procedure
Summary
The ability to precisely query functional hypotheses of protein function in cells requires the capacity to express rationally mutated proteins from genes under their native physiological regulation. Transgenes expressed in higher eukaryotes are derived from cDNAs, and lack native cis-regulatory elements or alternative splicing isoforms, often resulting in overexpression and deregulation artefacts. This may hinder proper phenotypic functional characterization of mutated genes, as well as the determination of the precise localization and interaction partners of the protein products. BAC transgenes modified to be RNAi resistant allow for conditional exposure of recessive mutations through selective depletion of endogenous protein, as an alternative to direct genome editing to overcome the limitations listed above (Bird et al, 2011; Bird and Hyman, 2008; Ding et al, 2009; Kittler et al, 2005; Rondelet et al, 2020; Scolz et al, 2012; Singh et al, 2020; Zheng et al, 2014)
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