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Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2013 Feb;30(1):9-12. doi: 10.3760/cma.j.issn.1003-9406.2013.01.003.

[Construction of wild-type and mutant SPAST vectors for the study of molecular mechanism of hereditary spastic paraplegia].

[Article in Chinese]

Author information

  • 1Department of Neurology, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.

Abstract

OBJECTIVE:

To construct wild-type and mutant pEGFP SPAST vectors and to explore the molecular mechanism of hereditary spastic paraplegia.

METHODS:

Mutant SPAST vector was constructed using overlap PCR method following construction of wild-type SPAST vector. Wild-type and mutant constructs were transfected to COS7 cells and subcellular localization of spastin was observed. Co-localizations of spastin and microtubule, spastin and mitochondria were viewed by immunofluorescence staining.

RESULTS:

Wild-type spastin is localized in plasma, and mutant spastin did not change its cellular localization. Wild-type and mutant spastins did not co-localize with microtubules and mitochondria by immunofluorescence analysis.

CONCLUSION:

Wild-type and mutant SPAST constructs were successfully generated. Mutant spastin did not change its localization in cells. Spastin does not co-localize with microtubules and mitochondria. This study may facilitate further studies on molecular mechanism of hereditary spastic paraplegia.

PMID:
23450470
[PubMed - indexed for MEDLINE]
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