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.
Article
in Chinese
| WPRIM
| ID: wpr-229808
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>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Physiology
/
Plasmids
/
Immunohistochemistry
/
Transfection
/
Cell Line
/
Blotting, Western
/
Reverse Transcriptase Polymerase Chain Reaction
/
RNA Interference
/
Alpha-Synuclein
/
Genetic Vectors
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Medical Genetics
Year:
2008
Type:
Article
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