FLP/FRT and Cre/lox recombination technology in C. elegans

Abstract

One of the most powerful aspects of biological inquiry using model organisms is the ability to control gene expression. A holy grail is both temporal and spatial control of the expression of specific gene products - that is, the ability to express or withhold the activity of genes or their products in specific cells at specific times. Ideally such a method would also regulate the precise levels of gene activity, and alterations would be reversible. The related goal of controlled or purposefully randomized expression of visible markers is also tremendously powerful. While not all of these feats have been accomplished in Caenorhabditis elegans to date, much progress has been made, and recent technologies put these goals within closer reach. Here, I present published examples of successful two-component site-specific recombination in C. elegans. These technologies are based on the principle of controlled intra-molecular excision or inversion of DNA sequences between defined sites, as driven by FLP or Cre recombinases. I discuss several prospects for future applications of this technology.

Keywords: Excision; Heat-shock; Inversion; Recombinase; Two-component system.

Figure 1

General strategies for excision, inversion and intersectional applications of Cre and FLP recombinase technologies.

Figure 2. Excision strategies

A. Figure 6A from Hoier et al., 2000 (Genes and Development) showing strategy to inactivate the apr-1(+) transgene in Pn.p cells. B. Figure 2A–C from Davis et al., 2008 (PLoS Genetics) showing a modular ORFeome and promoterome system to build FRT target plasmids. C. Figure 7A–C from Voutev and Hubbard 2008 (Genetics) showing strategies to build FRT target plasmids from Fire vectors or via Gateway, as well as single step Wormgate and ORFeome-based generation of RNAi hairpin-expressing constructs. D. Figure 2A (partial) from Macosko et al., 2009 (Nature) showing strategy for intersectional restricted expression of npr-1. Figures reproduced with permission.

Figure 3. Inversion strategies

A. Figure 2A (partial) from Vazques-Manrique et al., 2010 (Genomics) showing strategy for excision of integrated positive selection marker. B. Figure 3C from Flavell et al., 2013 (Cell) showing strategy for excision using nested lox sites. C. Figure 1B from Sohal et al., 2009 (Nature) showing events leading to stable excision using nested lox sites. Figures reproduced with permission.

Figure 4. Genome editing and recombination-mediated excision

A. Figure 4A, B from Lo et al., 2013 (Genetics) showing strategy for genomic excision of rex-32 sequences using precise genome editing to insert FRT sites. B. Figure 3A (partial) from Dickinson et al., 2013 (Nature Methods) showing strategy for excision of the unc-119(+) selectable marker. Figures reproduced with permission.