Activities of Various FLP Recombinases Expressed by Adenovirus Vectors in Mammalian Cells

Kondo, Shu; Takata, Yuki; Nakano, Masakazu; Saito, Izumu; Kanegae, Yumi

doi:10.1016/j.jmb.2009.04.057

Cited by 16 publications

(22 citation statements)

References 45 publications

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“…In addition, FLPe and FLPo might exhibit differential expression levels in different tissues. Surprisingly, a recent report by Kondo et al showed that FLPe exhibits lower recombination efficiency than the original wild type FLP on a molar basis even at 37°C [18]. However, the steady-state of FLPe protein level is 3 times of wild type FLP in mammalian cells, indicating that the higher recombination efficiency observed with FLPe is largely due to its enhanced thermostability in the expense of lower enzymatic activity.…”

Section: Discussionmentioning

confidence: 95%

High-Efficient FLPo Deleter Mice in C57BL/6J Background

Wang

Sun

et al. 2009

PLoS ONE

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Conditional gene manipulation in mice becomes a routine for genetic studies of mammalian gene functions. Additional site-specific recombinases such as FLP or φ31 provide one more level of gene manipulation flexibility. The recombination activity of the currently available FLP deleter mice remains low. We generated a new FLP deleter mouse line with the mouse codon-optimized FLPo gene in C57BJ/6 background, which showed superior recombination efficacy in comparison to FLPe deleter mice. 100% complete removal of FRT-flanked Neo cassette was observed in all F1 progeny mice carrying both FLPo and Neo cassette, which can be transmitted to F2 generation independent of FLPo activity. Our new FLPo transgenic mice (on pure C57BJ/6 background) will largely facilitate the gene targeting process and is valuable for conditional gene manipulation.

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Section: Discussionmentioning

confidence: 95%

High-Efficient FLPo Deleter Mice in C57BL/6J Background

Wang

Sun

et al. 2009

PLoS ONE

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“…Mice were generated using two independent, correctly targeted ESC clones assessed by PCR genotyping. Prior to generation of chimeras, the neomycin-resistance gene was removed by transient expression of hFLPe in ESCs, using pxCAhFLPe (Kondo et al, 2009) (a gift from Drs Yumi Kanegae and Izumu Saito, University of Tokyo, Japan). The heterozygous mice were backcrossed at least six times to C57BL/6J mice before being used for this study.…”

Section: Micementioning

confidence: 99%

FGF2-induced Ras/Erk MAPK signalling maintains lymphatic endothelial cell identity by up-regulating endothelial cell-specific gene expression and suppressing TGFβ signalling via Smad2

Ichise

Yoshida

Ichise

2013

Journal of Cell Science

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The lymphatic endothelial cell (LEC) fate decision program during development has been described. However, the mechanism underlying the maintenance of differentiated LEC identity remains largely unknown. Here, we show that fibroblast growth factor 2 (FGF2) plays a fundamental role in maintaining a differentiated LEC trait. In addition to demonstrating the appearance of LECs expressing a-smooth muscle actin in mouse lymphedematous skin in vivo, we found that mouse immortalised LECs lose their characteristics and undergo endothelial-to-mesenchymal transition (EndMT) when cultured in FGF2-depleted medium. FGF2 depletion acted synergistically with transforming growth factor (TGF) b to induce EndMT. We also found that H-Ras-overexpressing LECs were resistant to EndMT. Activation of H-Ras not only upregulated FGF2-induced activation of the Erk mitogen activated protein kinases (MAPK3 and MAPK1), but also suppressed TGFb-induced activation of Smad2 by modulating Smad2 phosphorylation by MAPKs. These results suggest that FGF2 regulates LEC-specific gene expression and suppresses TGFb signalling in LECs through Smad2 in a Ras-MAPK-dependent manner. Taken together, our findings provide a new insight into the FGF2-Ras-MAPK-dependent mechanism that maintains and modulates the LEC trait.

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“…Nevertheless, ZmU-BI1:FLPe expression in the gametophyte is clearly not sufficient for marker excision, and the successful use of FLPe would require improving its efficiency or using proper gametophytic promoters. A quantitative comparison of FLPwt and FLPe activities in mammalian cells found that the recombination efficiency of FLPe on a molar basis was not higher than that of FLPwt, even at 37°C (Kondo et al 2009). Therefore, higher efficiency of FLPe in plant or animal cells is due not to higher enzyme activity but to the higher steady-state level of the FLPe protein, owing to its thermo-stability.…”

Section: Discussionmentioning

confidence: 99%

Strong activity of FLPe recombinase in rice plants does not correlate with the transmission of the recombined locus to the progeny

Nguyen¹,

Underwood

Nandy

et al. 2014

Plant Biotechnol Rep

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Efficient methods for DNA excision are needed for removing selectable marker genes from transgenic plants. The present work evaluated the enhanced FLP recombinase, FLPe, for excising FLP recombination target (FRT)-flanked marker genes, and generating marker-free rice lines. Previously, the transient FLPe activity was found to be at least threefold higher on the transgene locus compared to that of FLPwt, the wild-type FLP recombinase. In this study, transgenic plants expressing FLPe were cross-pollinated with the plants harboring FRT site to analyze marker excision in F1 plants, and the transmission of marker-free locus to F2 progeny. The FLPe activity, expressed by the strong promoter (maize ubiquitin-1 gene), efficiently excised FRT-flanked marker gene in rice plants. However, marker excision in F2 progeny was tightly linked with the presence of FLPe gene, suggesting insufficient recombination in the gametophyte. The maize ubiquitin-1 promoter is reportedly active in gametophytic tissue and effective in meiotic transmission of the marker-free locus generated by Cre-lox recombination. Therefore, the observed lack of meiotic transmission in this study is possibly due to the limited efficiency of FLPe recombinase. While the reason for the FLPe inefficiency in the gametophyte is not clear, this work highlights the constraints of FLPe recombinase in generating stable marker-free plant lines through cross-pollination or gene induction methods.

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Activities of Various FLP Recombinases Expressed by Adenovirus Vectors in Mammalian Cells

Cited by 16 publications

References 45 publications

High-Efficient FLPo Deleter Mice in C57BL/6J Background

High-Efficient FLPo Deleter Mice in C57BL/6J Background

FGF2-induced Ras/Erk MAPK signalling maintains lymphatic endothelial cell identity by up-regulating endothelial cell-specific gene expression and suppressing TGFβ signalling via Smad2

Strong activity of FLPe recombinase in rice plants does not correlate with the transmission of the recombined locus to the progeny

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