June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Identification of heterotypic rox sites for use in novel Dre recombination strategies
Author Affiliations & Notes
  • Eileen Nguyen
    Retinal Circuit Development and Genetics Unit, National Eye Institute, Bethesda, MD
  • Katherine Chuang
    Retinal Circuit Development and Genetics Unit, National Eye Institute, Bethesda, MD
  • Yuri V Sergeev
    OGVFB, National Eye Institute, Bethesda, MD
  • Tudor Badea
    Retinal Circuit Development and Genetics Unit, National Eye Institute, Bethesda, MD
  • Footnotes
    Commercial Relationships Eileen Nguyen, None; Katherine Chuang, None; Yuri Sergeev, None; Tudor Badea, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4664. doi:
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      Eileen Nguyen, Katherine Chuang, Yuri V Sergeev, Tudor Badea; Identification of heterotypic rox sites for use in novel Dre recombination strategies. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4664.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The widely used Cre-loxP recombination system comprises an extensive collection of mouse lines expressing Cre or Cre-dependent targets and flexibility of strategies used to generate tightly regulated or combinatorial recombined target sites. Dre, a Cre homologue has similar recombination efficiency and specificity for its own target, roxP, sites, while exhibiting no cross reactivity with either Cre or Flp. Dre has been successfully used in genetic manipulations, alone or in combination with other recombinases in mouse and zebrafish models. However, little is known about the structural and functional Dre protein-roxP DNA interaction. We now report a substitution screen aimed at identifying roxP base pairs critical for self-recognition.

Methods: Degenerate roxP sequence libraries were tested for Dre recombination using a bacterial antibiotic resistance screen. Recombination specificity and efficiency of novel sites was confirmed in bacterial recombination assays. Furthermore, eukaryotic cells (HEK293) were transfected with roxP-dependent cassettes containing fluorescent reporters to determine their recombination efficiencies with themselves and their incompatibility with the wild type sequence.

Results: These novel sites self-recombine with high efficiency in prokaryotic and eukaryotic cells, but do not recombine with the wild type sequence. We confirmed wild type incompatibility for the seven most encountered heterotypic spacers, and efficient self-recombination for five. Our findings are consistent with the Cre - loxP system, in which equivalent substitutions had strong effects on compatibility and only mild effects on self-recombination. Recombination specificity and efficiency were also conserved in eukaryotic cells, allowing for the development of a tight inversion - excision strategy, which we call FREX.

Conclusions: We report the identification of several roxP variants that are incompatible with the wild type Dre recombinase roxP target site consensus, but recombine efficiently with themselves. Since our constructs had zero recombination with the wild type and were not targeted by the Cre recombinase, they would be ideal in future combinatorial genetic manipulations involving Dre.

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