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[Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2008 Apr; 25(2):128-31.ZY

Abstract

OBJECTIVE

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

Authors+Show Affiliations

Chen T
Department of Neurology, the Affiliated Xiangya Hospital, Central South University, Changsha, Hunan, 410008 P. R. China.
Tang BS
No affiliation info available
Liao XP
No affiliation info available

MeSH

Blotting, WesternCell LineGenetic VectorsHumansImmunohistochemistryPlasmidsRNA InterferenceReverse Transcriptase Polymerase Chain ReactionTransfectionalpha-Synuclein

Pub Type(s)

English Abstract
Journal Article

Language

chi

PubMed ID

18393229

Citation

Chen, Tao, et al. "[Blockade of the Aberrant Aggregation of Alpha-synuclein in HEK293 Cells Induced By Overexpression of Wild-type Alpha-synuclein By RNA Interference]." Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, vol. 25, no. 2, 2008, pp. 128-31.
Chen T, Tang BS, Liao XP. [Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2008;25(2):128-31.
Chen, T., Tang, B. S., & Liao, X. P. (2008). [Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, 25(2), 128-31.
Chen T, Tang BS, Liao XP. [Blockade of the Aberrant Aggregation of Alpha-synuclein in HEK293 Cells Induced By Overexpression of Wild-type Alpha-synuclein By RNA Interference]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2008;25(2):128-31. PubMed PMID: 18393229.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference]. AU - Chen,Tao, AU - Tang,Bei-sha, AU - Liao,Xiao-ping, PY - 2008/4/9/pubmed PY - 2009/3/20/medline PY - 2008/4/9/entrez SP - 128 EP - 31 JF - Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics JO - Zhonghua Yi Xue Yi Chuan Xue Za Zhi VL - 25 IS - 2 N2 - OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. SN - 1003-9406 UR - https://www.unboundmedicine.com/medline/citation/18393229/[Blockade_of_the_aberrant_aggregation_of_alpha_synuclein_in_HEK293_cells_induced_by_overexpression_of_wild_type_alpha_synuclein_by_RNA_interference]_ DB - PRIME DP - Unbound Medicine ER -