High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells

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.

Figure 1. Recombination analysis of SSR activity on stably transfected ES cells.

(A) Schematic diagram of SSR reporter assay system. A ΦC31 β-galactosidase reporter ES cell line was constructed at the ROSA26 locus , except that the stop cassette was flanked by minimal 35 bp attB and 39 bp attP target DNA recognition sites. These ES cells were used to generate a ΦC31 reporter mouse strain. (B) ES reporter cell lines for Cre , FLP , and ΦC31 (described within) were stably co-transfected with linearized SSR and PGKHygromycin. SSR-mediated recombination activity was assessed on hygromycin-resistant ES cell colonies by X-gal (Cre and ΦC31) and AP staining (FLP). Variable degrees of recombination were observed in colonies, as judged by the percentage of cells reacting with X-Gal; representative ES cell colonies are shown. N.D.- not detected.

Figure 2. Analysis of R26ΦC31 and R26ΦC31o activity in vivo.

(A) Diagram of ΦC31 and ΦC31o knock-in vectors targeted to the ROSA26 locus. Whole mount X-Gal staining of E10.5 (B) R26ΦC31; R26attR and (C) R26ΦC31o; R26attR compound heterozygous embryos. (D) Sagittal section of X-Gal stained E10.5 R26ΦC31o; R26attR compound heterozygote.