Construction of wild-type and mutant SPAST vectors for the study of molecular mechanism of hereditary spastic paraplegia / 中华医学遗传学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct wild-type and mutant pEGFP SPAST vectors and to explore the molecular mechanism of hereditary spastic paraplegia.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>