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      Construction of engineered murine Embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP

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          Abstract

          Objective: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. Methods: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. Results: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR. Conclusion: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.

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          Most cited references42

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          FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease.

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            Receptor specificity of the fibroblast growth factor family.

            Fibroblast growth factors (FGFs) are essential molecules for mammalian development. The nine known FGF ligands and the four signaling FGF receptors (and their alternatively spliced variants) are expressed in specific spatial and temporal patterns. The activity of this signaling pathway is regulated by ligand binding specificity, heparan sulfate proteoglycans, and the differential signaling capacity of individual FGF receptors. To determine potentially relevant ligand-receptor pairs we have engineered mitogenically responsive cell lines expressing the major splice variants of all the known FGF receptors. We have assayed the mitogenic activity of the nine known FGF ligands on these cell lines. These studies demonstrate that FGF 1 is the only FGF that can activate all FGF receptor splice variants. Using FGF 1 as an internal standard we have determined the relative activity of all the other members of the FGF family. These data should serve as a biochemical foundation for determining developmental, physiological, and pathophysiological processes that involve FGF signaling pathways.
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              Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction.

              FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, we have mutated FGFR2 by deleting the entire immunoglobin-like domain III of the receptor. We showed that murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2(DeltaIgIII/DeltaIgIII) embryos displayed two distinct defects that resulted in failures in formation of a functional placenta. About one third of the mutants failed to form the chorioallantoic fusion junction and the remaining mutants did not have the labyrinthine portion of the placenta. Consequently, all mutants died at 10-11 days of gestation. Interestingly, Fgfr2(DeltaIgIII/DeltaIgIII) embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction.
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                Author and article information

                Journal
                biocell
                Biocell
                Biocell
                Sociedad Latinoamericana de Microscopía Electrónica.; Centro Regional de Investigaciones Científicas y Tecnológicas (Mendoza, Argentina) (Mendoza, Mendoza, Argentina )
                0327-9545
                1667-5746
                August 2006
                : 30
                : 2
                : 269-278
                Affiliations
                [01] Chongqing orgnameThe Third Military Medical University orgdiv1Daping Hospital orgdiv2Laboratory of trauma center China
                Article
                S0327-95452006000200002 S0327-9545(06)03000200002
                312a1f19-80fc-4f7d-9545-4a847a06ee6a

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                History
                : 29 January 2005
                : 30 May 2006
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 25, Pages: 10
                Product

                SciELO Argentina


                Cre,Fibroblast growth factor receptor-2 (FGFR2),ES cells,Homologous recombination,Conditional knockout

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