High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells - PubMed (original) (raw)

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

Christopher S Raymond et al. PLoS One. 2007.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

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

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.

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