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Establishment of human neuroblastoma SH-SY5Y cell line stably silencing beclin1 / 生物医学工程学杂志
Journal of Biomedical Engineering ; (6): 1085-1089, 2014.
Artículo en Chino | WPRIM | ID: wpr-234453
ABSTRACT
The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E. coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28% ± 1.45% (P < 0.05)and 75.50% ± 2.63% (P < 0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SYSY cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.
Asunto(s)
Texto completo: Disponible Índice: WPRIM (Pacífico Occidental) Asunto principal: Plásmidos / ARN Mensajero / Transfección / Regulación hacia Abajo / Silenciador del Gen / ARN Interferente Pequeño / Línea Celular Tumoral / Escherichia coli / Proteínas Reguladoras de la Apoptosis / Beclina-1 Tipo de estudio: Estudio pronóstico Límite: Humanos Idioma: Chino Revista: Journal of Biomedical Engineering Año: 2014 Tipo del documento: Artículo

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Texto completo: Disponible Índice: WPRIM (Pacífico Occidental) Asunto principal: Plásmidos / ARN Mensajero / Transfección / Regulación hacia Abajo / Silenciador del Gen / ARN Interferente Pequeño / Línea Celular Tumoral / Escherichia coli / Proteínas Reguladoras de la Apoptosis / Beclina-1 Tipo de estudio: Estudio pronóstico Límite: Humanos Idioma: Chino Revista: Journal of Biomedical Engineering Año: 2014 Tipo del documento: Artículo