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.
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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