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

Significance

Tools that afford spatiotemporal control of gene expression are crucial for studying genes and processes in multicellular organisms. Stop cassettes consist of exogenous sequences that interrupt gene expression and flanking site-specific recombinase sites to allow for tissue-specific excision and restoration of function by expression of the cognate recombinase. We describe a stop cassette called a flexon, composed of an artificial exon flanked by artificial introns that can be flexibly positioned in a gene. We demonstrate its efficacy 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 diverse areas including development, neuroscience, and metabolism.

Abstract

Conditional gene expression is a powerful tool for 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 lox sites into the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been successfully used in many experimental systems, this design has certain 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 termination of translation and nonsense-mediated decay of the mRNA and allowing for flexible placement into a gene. We demonstrate its efficacy in Caenorhabditis elegans by showing that, when promoters that cause weak 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 lineages. We also demonstrate its efficacy in an endogenous gene context: we inserted a flexon into the Argonaute gene rde-1 to abrogate RNA interference (RNAi), and restored RNAi tissue specifically by expression of Cre. 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 may be applied.

Footnotes

    • Accepted December 4, 2021.
  • Author contributions: J.M.S. designed research; J.M.S. performed research; J.M.S. analyzed data; J.M.S. and I.G. wrote the paper; and I.G. supervised research.

  • Reviewers: J.K., Johns Hopkins University; B.G., Rutgers The State University of New Jersey; and A.R., University of Minnesota Twin Cities.

  • The authors declare no competing interest.

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

Data Availability

All study data are included in the article and/or SI Appendix.

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