Raymond 2007 FLPo WC31o

Raymond et al. (2007). PLoS ONE DOI: 10.1371/journal.pone.0000162 Citations: 403

Only Cre achieved efficient recombination in mammalian cells, with FLP and phiC31 inefficient. This paper synthesized mouse codon-optimized FLPo and phiC31o de novo. Both matched Cre's efficiency in ES cells, and phiC31o gave broad embryo staining versus mosaic native phiC31.

Parts used

FlpoSV40 NLSKozak Sequence

Key findings

  • Mouse codon-optimized FLPo and PhiC31o recombinases were synthesized de novo using GeneOptimizer algorithm with modifications including mouse codon usage, removal of cryptic splice sites and TATA boxes, reduced CpG dinucleotides in PhiC31o to prevent methylation-induced silencing, and modified G/C content in FLPo to prolong mRNA half-life. Both genes incorporated dual stop codons, Kozak consensus sequences, and SV40 nuclear localization signals under PGK promoter control.
  • Stable transfection assays in ES reporter cell lines demonstrated that FLPo and PhiC31o achieved recombination efficiencies similar to Cre recombinase, with majority of colonies showing complete alkaline phosphatase or X-gal staining. Native FLPe showed no detectable recombination while native PhiC31 exhibited mosaic or absent X-gal staining, representing improvements of >20-fold for FLPo and substantial enhancement for PhiC31o.
  • In vivo validation using ROSA26-targeted strains showed that R26-PhiC31o; R26attR compound heterozygous E10.5 embryos displayed broad X-gal staining throughout the embryo, compared to variable low-level mosaic staining in R26-PhiC31; R26attR embryos. R26-PhiC31 and R26-PhiC31o heterozygous and homozygous mice were viable without deleterious effects from widespread recombinase expression.

Abstract

DNA site-specific recombinases (SSRs) such as Cre, FLPe, and phiC31, are powerful tools for analyzing gene function in vertebrates. While the availability of multiple high-efficiency SSRs would facilitate a wide array of genomic engineering possibilities, efficient recombination in mammalian cells has only been observed with Cre recombinase. Here we report the de novo synthesis of mouse codon-optimized FLP (FLPo) and PhiC31 (PhiC31o) SSRs, which result in recombination efficiencies similar to Cre.