title: [Establishment of human neuroblastoma SH-SY5Y cell line stably silencing beclin1].
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.
authors.forename: Chuanjie
authors.initials: C
authors.lastname: Wei
authors.forename: Shuang
authors.initials: S
authors.lastname: Xiao
authors.forename: Lan
authors.initials: L
authors.lastname: Jiang
authors.forename: Yan
authors.initials: Y
authors.lastname: Tan
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
authors.forename: Dingxin
authors.initials: D
authors.lastname: Long
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D000071186:Beclin-1; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004926:Escherichia coli; D020868:Gene Silencing; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009447:Neuroblastoma; D010957:Plasmids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering