[Construction and analysis of recombinant eukaryotic expression plasmids for SOX10, the causative gene of Warrdenburg syndrome].

OBJECTIVE

To study the exogenous expression and subcellular localization of wild type (WT) and mutant SOX10 proteins in vitro through generation of expression plasmids in order to reveal the pathogenesis of Waardenburg syndrome (WS).

METHODS

The plasmids pECE-SOX10 and pCMV-Flag were ligated after they were subjected to double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-SOX10-Flag, which was as a template to generate expression plasmids for novel mutations G37fs, G38fs and E248fs of the SOX10 gene. The constructs were verified by direct sequencing. NIH3T3 cells were transiently transfected with the expression plasmids of wide type SOX10, G37fs, G38fs and E248fs, respectively. The exogenous expression of WT SOX10 protein and mutant G37fs, G38fs and E248fs proteins were analyzed using Western blot assay, while their subcellular distribution were observed with an immunofluorescence assay.

RESULTS

The DNA sequences of expression plasmids for SOX10 and its mutant G37fs, G38fs and E248f were all correct. Both WT and mutant SOX10 proteins were detected at the expected site. WT SOX10 and E248fs proteins have only localized in the nucleus, whereas G37fs and G38fs proteins showed aberrant localization in both cytoplasm and nucleus.

CONCLUSION

Recombinant eukaryotic expression plasmids for the SOX10 gene and its mutants were successfully constructed. Preliminary analysis showed that the mutations have affected the subcellular distribution of WT SOX10 proteins, which has laid a basis for further study of the molecular mechanism of WS caused by SOX10 gene mutations.