Assuntos
Animais , Masculino , Camundongos , /fisiologia , /metabolismo , Proteína do Retinoblastoma/fisiologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , /metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , beta-Galactosidase/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Camundongos Knockout , Nefropatias/genética , Nefropatias/patologia , Transdução de Sinais/fisiologiaAssuntos
Animais , Masculino , Camundongos , Genes p53/fisiologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/fisiologia , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismo , Nefropatias/genética , Nefropatias/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais/fisiologiaRESUMO
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 andconfirmed by PCR and digesti on 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.
Assuntos
Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Estruturas Embrionárias/citologia , Marcação de Genes , Genoma/genética , Vetores Genéticos/genética , Sequência de Bases , Integrases/genética , Integrases/metabolismo , Dados de Sequência Molecular , Recombinação Genética , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
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 andconfirmed by PCR and digesti on 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.(AU)
