Dong-Mei Liu1, Ling Jin, Hao Wang, Huan-Ying Zhao, Chun-Li Zhao, Hui Yang. 1. Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing 100069, China.
Abstract
OBJECTIVE: To silence the expression of alpha-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability. METHODS: We identified two 19-nucleotide stretches within the coding region of the alpha-synuclein gene and designed three sets of oligonucleotides to generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR/H1/TO vector and transfected into MN9D dopaminergic cells. alpha-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT). RESULTS: The mRNA and protein levels of alpha-synuclein gene were significantly down-regulated in pSH2/alpha-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/alpha-SYN-transfected cells showed no significant difference. Silencing alpha-synuclein expression does not affect cell proliferation but may decrease cell viability. CONCLUSION: Our results demonstrated pSH2/alpha-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse alpha-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of alpha-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. alpha-Synuclein may be important for the viability of MN9D cells, and loss of alpha-synuclein may induce cell injury directly or indirectly.
OBJECTIVE:To silence the expression of alpha-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability. METHODS: We identified two 19-nucleotide stretches within the coding region of the alpha-synuclein gene and designed three sets of oligonucleotidesto generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR/H1/TO vector and transfected into MN9D dopaminergic cells. alpha-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT). RESULTS: The mRNA and protein levels of alpha-synuclein gene were significantly down-regulated in pSH2/alpha-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/alpha-SYN-transfected cells showed no significant difference. Silencing alpha-synuclein expression does not affect cell proliferation but may decrease cell viability. CONCLUSION: Our results demonstrated pSH2/alpha-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mousealpha-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of alpha-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. alpha-Synuclein may be important for the viability of MN9D cells, and loss of alpha-synuclein may induce cell injury directly or indirectly.
Authors: Patrick J Paddison; Amy A Caudy; Emily Bernstein; Gregory J Hannon; Douglas S Conklin Journal: Genes Dev Date: 2002-04-15 Impact factor: 11.361
Authors: Brian Spencer; Ivy Trinh; Edward Rockenstein; Michael Mante; Jazmin Florio; Anthony Adame; Omar M A El-Agnaf; Changyoun Kim; Eliezer Masliah; Robert A Rissman Journal: Neurobiol Dis Date: 2019-03-05 Impact factor: 5.996