Floxed exon (Flexon): a flexibly positioned stop cassette for recombinase-mediated conditional gene expression

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Importance

Tools that allow spatio-temporal control of gene expression are crucial for the study of genes and processes in multicellular organisms. Stop cassettes consist of exogenous sequences that shut down gene expression and site-specific recombinase flanking sites to allow tissue-specific excision and restoration of function through cognate recombinase expression. We describe a stop tape called flex, composed of an artificial exon flanked by artificial introns that can be flexibly positioned in a gene. We demonstrate its effectiveness in Caenorhabditis elegans for lineage-specific control of gene expression and for tissue-specific RNA interference and discuss other potential uses. The Flexon approach should be feasible in any system amenable to site-specific recombination-based methods and applicable to various fields including developmental, neuroscience, and metabolism.

Summary

Conditional gene expression is a powerful tool for the genetic analysis of biological phenomena. In the widely used “lox-stop-lox” approach, insertion of a stop cassette consisting of a series of stop codons and polyadenylation signals flanked by smoked salmon sites in the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of the Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been used successfully in many experimental systems, this design has some limitations. Here we describe the Floxed Exon (Flexon) approach, which uses a stop cassette composed of an artificial exon flanked by artificial introns, designed to cause premature translational termination and nonsense decay. of mRNA and allowing flexible placement within a gene. We demonstrate its effectiveness in Caenorhabditis elegans by showing that when promoters that cause low and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression of green fluorescent protein (GFP) is obtained in specific lines. We also demonstrate its effectiveness in an endogenous gene context: we inserted a flex in the Argonaute gene rde-1 to abrogate RNA interference (RNAi) and restore RNAi tissue specifically through Cre expression. Finally, we describe several potential additional applications of the Flexon approach, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation, and generation of genetic mosaics. The Flexon approach should be feasible in any system where a site-specific recombination-based method can be applied.

Footnotes

    • Accepted December 4, 2021.
  • Author contributions: research designed by JMS; JMS has done research; JMS analyzed the data; JMS and IG authored the article; and IG-supervised research.

  • Reviewers: JK, Johns Hopkins University; BG, Rutgers The State University of New Jersey; and AR, University of Minnesota Twin Cities.

  • The authors declare no competing interests.

  • This article contains additional information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2117451119/-/DCSupplemental

Data availability

All study data is included in the article and/or IS Annex.

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