Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics · 2013
[Construction of wild-type and mutant SPAST vectors for the study of molecular mechanism of hereditary spastic paraplegia].
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Yan Ya-ping, Pu Jia-li, Zhang Bao-rong, Zhao Guo-hua
Аннотация
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.
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.