Establishment of human neuroblastoma SH-SY5Y cell line stably silencing beclin1.
- Author:
Chuanjie WEI
;
Shuang XIAO
;
Lan JIANG
;
Yan TAN
;
Bo HUANG
;
Dingxin LONG
- Publication Type:Journal Article
- MeSH:
Apoptosis Regulatory Proteins;
genetics;
metabolism;
Beclin-1;
Cell Line, Tumor;
Down-Regulation;
Escherichia coli;
Gene Silencing;
Humans;
Membrane Proteins;
genetics;
metabolism;
Neuroblastoma;
Plasmids;
RNA, Messenger;
RNA, Small Interfering;
Transfection
- From:
Journal of Biomedical Engineering
2014;31(5):1085-1089
- CountryChina
- Language:Chinese
-
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.
