Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference

Coumoul, Xavier; Shukla, Vivek; Li, Cuiling; Wang, Rui-Hong; Deng, Chu‐Xia

doi:10.1093/nar/gni100

Cited by 85 publications

(82 citation statements)

References 40 publications

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“…This most likely reflects a reduced efficiency where transient transfection of two plasmid components is necessary. In summary, our results support the conclusion of others on the efficiency of conditional regulatory components embedded between the PSE and DSE within the U6 promoter (Coumoul et al, 2004(Coumoul et al, , 2005, and extend these findings by screening for an optimal placement.…”

supporting

confidence: 91%

“…Cre recombinase has played the central role in current methodologies to generate conditionally active alleles in the mouse; the large number of tissuespecific Cre mouse lines are a tremendous resource for functional genetic studies. Various schemes for Cremediated conditional expression of shRNA have been tested with conflicting conclusions as to the ''neutrality'' of embedded components within the regulatory region of the driver pol III promoter (Tiscornia et al, 2004;Ventura et al, 2004;Coumoul et al, 2004Coumoul et al, , 2005.…”

mentioning

confidence: 99%

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Reproducible and inducible knockdown of gene expression in mice

McMahon

2006

Genesis

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Summary: RNA interference (RNAi) has emerged as an efficient approach for rapid analysis of gene function. In mammalian cells, vector-based expression of small hairpin RNAs (shRNA) produces potent and stable gene knockdown effects. An inducible RNAi system with reproducible levels of siRNA expression will extend the usefulness of this methodology to the identification of gene functions within the developing or adult mouse. We present evidence that an RNA polymerase III-driven U6 promoter with stuffer sequences flanked by loxP sites inserted at three different sites within the promoter drives shRNA expression in a Cre recombinase-dependent manner. We utilized this approach to develop a generic strategy for the reproducible knockdown of gene expression in mice. By placing the inducible shRNA cassette into the ROSA26 locus of the mouse, we were able to generate reproducible levels of controlled expression of shRNA to produce discernable phenotypes in vitro and in vivo. This approach circumvents the prescreening of random integration in embryonic stem cell clones and further enables conditional gene knockdown with temporal and/or tissue specificity. This methodology should expedite large-scale functional studies.

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supporting

confidence: 91%

mentioning

confidence: 99%

Reproducible and inducible knockdown of gene expression in mice

McMahon

2006

Genesis

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“…Conditional inactivation of Fgfr1 and Fgfr2 in limb bud mesenchyme (using Prx1-Cre) or inactivation of Fgfr1 in all mesenchyme [using T (brachyury)-Cre] results in severe skeletal hypoplasia (Verheyden et al 2005;Yu and Ornitz 2008). In contrast, inactivation of Fgfr1 or Fgfr2 in distal limb bud mesenchyme (using AP2-Cre) results in considerably milder skeletal patterning phenotypes (Coumoul et al 2005;Li et al 2005). These experiments collectively demonstrate redundancy between FGFR1 and FGFR2 in distal limb mesenchyme.…”

Section: Fgf Signaling During Skeletal Developmentmentioning

confidence: 72%

Fibroblast growth factor signaling in skeletal development and disease

2015

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Fibroblast growth factor (FGF) signaling pathways are essential regulators of vertebrate skeletal development. FGF signaling regulates development of the limb bud and formation of the mesenchymal condensation and has key roles in regulating chondrogenesis, osteogenesis, and bone and mineral homeostasis. This review updates our review on FGFs in skeletal development published in Genes & Development in 2002, examines progress made on understanding the functions of the FGF signaling pathway during critical stages of skeletogenesis, and explores the mechanisms by which mutations in FGF signaling molecules cause skeletal malformations in humans. Links between FGF signaling pathways and other interacting pathways that are critical for skeletal development and could be exploited to treat genetic diseases and repair bone are also explored.

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“…These results suggest that AER initiation requires ectodermal FGFR2 function to respond to mesenchymal FGF10 signaling, as mice lacking mesenchymal Fgf10 are also limbless (Min et al, 1998;Sekine et al, 1999). However, mesenchymal expression of FGFR2 (Coumoul et al, 2005), as well as of FGFR1 Verheyden et al, 2005), is essential for skeletal progenitor cells to respond to AER-FGFs to ensure normal skeletal formation and patterning. In this study, we…”

Section: Introductionmentioning

confidence: 91%

The apical ectodermal ridge is a timer for generating distal limb progenitors

Perdue

et al. 2008

Development

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The apical ectodermal ridge (AER) is a transient embryonic structure essential for the induction, patterning and outgrowth of the vertebrate limb. However, the mechanism of AER function in limb skeletal patterning has remained unclear. In this study, we genetically ablated the AER by conditionally removing FGFR2 function and found that distal limb development failed in mutant mice. We showed that FGFR2 promotes survival of AER cells and interacts with Wnt/␤-catenin signaling during AER maintenance. Interestingly, cell proliferation and survival were not significantly reduced in the distal mesenchyme of mutant limb buds. We established Hoxa13 expression as an early marker of distal limb progenitors and discovered a dynamic morphogenetic process of distal limb development. We found that premature AER loss in mutant limb buds delayed generation of autopod progenitors, which in turn failed to reach a threshold number required to form a normal autopod. Taken together, we have uncovered a novel mechanism, whereby the AER regulates the number of autopod progenitors by determining the onset of their generation.

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Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference

Cited by 85 publications

References 40 publications

Reproducible and inducible knockdown of gene expression in mice

Reproducible and inducible knockdown of gene expression in mice

Fibroblast growth factor signaling in skeletal development and disease

The apical ectodermal ridge is a timer for generating distal limb progenitors

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