Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference / 中华医学遗传学杂志
Chinese Journal of Medical Genetics
; (6): 128-131, 2008.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-229808
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To construct specific and effective RNA interference(RNAi) plasmid for alpha-synuclein gene and investigate RNAi blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein.</p><p><b>METHODS</b>Hairpin RNAs for four target sites were designed to construct four RNAi plasmids pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4, using plasmid pBSHH1 vector under the control of the H1 promoter. Western blot and reverse transcription-PCR(RT-PCR) were performed to screen the most specific and effective RNAi plasmid. After confirming the sequences of the plasmids, they were co-transfected into HEK293 cells with the recombinant plasmids alpha-synuclein-pEGFP by using lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence and immunocytochemistry for alpha-synuclein. The inclusions in the cultured cells were identified with HE staining.</p><p><b>RESULTS</b>By Western blot and RT-PCR, pSYNi-1 showed the most effective RNAi gene silencing effect (69.6%). After transfecting the RNAi plasmid, the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein was inhibited. The Lewy body-like inclusions were found in cytoplasm of cultured cells in control group, but disappeared in HEK293 cells cotransfected by pSYNi-1 and alpha-synuclein-pEGFP plasmid.</p><p><b>CONCLUSION</b>RNAi can block the aberrant aggregation and Lewy body-like inclusion formation in cytoplasm of HEK293 cell induced by overexpression of wild-type alpha-synuclein.</p>
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Fisiologia
/
Plasmídeos
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Imuno-Histoquímica
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Transfecção
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Linhagem Celular
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Western Blotting
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
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Interferência de RNA
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Alfa-Sinucleína
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Vetores Genéticos
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Medical Genetics
Ano de publicação:
2008
Tipo de documento:
Artigo