Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

Jung, Ui-Jung; Park, Sun; Lee, Gwang; Shin, Ho‐Joon; Kwon, Myung-Hee

doi:10.1016/j.bbrc.2007.08.145

Cited by 3 publications

(3 citation statements)

References 15 publications

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“…Six variants had guanine nucleotides at positions 2 and 7 in the spacer ( Figure 2D ), while a seventh had cytosine nucleotides on either side (rox8), in keeping with the noncompatible consensus derived from our screen (Ag/cGCCggT, Figure 2D ). In fact, similar observations have been reported in screens for loxP-incompatible lox sites ( Lee and Saito 1998 ; Siegel et al 2001 ; Langer et al 2002 ; Missirlis et al 2006 ; Jung et al 2007 ), in which self-compatible, efficiently recombining sequences tend to prefer G or C at positions 2 and 7 ( Langer et al 2002 ; Missirlis et al 2006 ), and the general recombination efficiency is associated with the presence of G bases ( Missirlis et al 2006 ). However, it should be pointed out that in the saturation screen performed in Missirlis et al (2006 ), the spacers exhibiting the highest frequency of self-recombination also had a high degree of promiscuity Figure 5 in ( Missirlis et al 2006 ), but that most of that promiscuity may be with other G containing spacers, since sequences containing Gs at positions 2 and 7 were less likely to recombine with spacers containing other nucleotides at those positions, Figure 6 in ( Missirlis et al 2006 ).…”

Section: Discussionsupporting

confidence: 76%

“…loxP strand breaks occur between spacer positions 1–2 on the top strand and 7–8 on the bottom strand ( Figure 1A , vertical arrows) ( Hoess and Abremski 1985 ; Guo et al 1997 ). Several screens have been performed to explore the influence of the 8 bp spacer sequence on recombination efficiency and specificity ( Lee and Saito 1998 ; Siegel et al 2001 ; Langer et al 2002 ; Missirlis et al 2006 ; Jung et al 2007 ). Systematic base substitution analysis showed that single mutations of the loxP spacer at position 7, or double mutants involving position 7 plus any of positions 2–5 ( e.g.…”

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confidence: 99%

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Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

Chuang

Nguyen

Sergeev

et al. 2016

G3 Genes|Genomes|Genetics

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Site-specific recombinases (SSRs) such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.

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Section: Discussionsupporting

confidence: 76%

mentioning

confidence: 99%

Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

Chuang

Nguyen

Sergeev

et al. 2016

G3 Genes|Genomes|Genetics

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show abstract

“…Under some in vivo conditions, Cre will carry out recombination between incompatible sites and will carry out deletions for sites arranged as inverted repeats at a measurable frequency (74). In some cases, the sequences of the recombinant products cannot be explained by Cre-mediated strand exchange of heterologous crossovers, suggesting that cellular factors may play a role in a subset of these noncanonical rearrangements (75).…”

Section: Requirements For Loxp Homologymentioning

confidence: 99%

Cre Recombinase

Duyne

2015

Microbiol Spectr

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The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

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Cre Recombinase

Duyne

2015

Mobile DNA III

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Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

Cited by 3 publications

References 15 publications

Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

Cre Recombinase

Cre Recombinase

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