A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

Abstract

ABSTRACTConditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established “lox-stop-lox” approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5′ untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. 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 is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.Summary statementThe Floxed exon (Flexon), a stop cassette that can be inserted flexibly into the coding region of any gene, facilitates Cre-mediated conditional gene expression.