Analysis of the expression of normal and mutant full⁃length atrophin⁃1 gene in stable and transient transfected eukaryotic cell line

Abstract

Objective To establish eukaryotic expression system of full ⁃ length gene in normal [CAG repeats (Q19)] and abnormal [CAG repeats (Q82 and Q66)] atrophin ⁃ 1 [the pathogenic gene of dentatorubral⁃pallidoluysian atrophy (DRPLA)]. To compare the expression system of stable and transient transfected cell line by observing the cellular appearance and atrophin⁃1 fusion protein expression and its distribution for further research on the establishment of a fine cell model. Methods Based on the 2 established stable transfected green⁃fluoresent protein (GFP)⁃atrophin⁃1⁃Q19/Flp⁃In TREx293 and GFP⁃ atrophin ⁃ 1 ⁃ Q82/Flp ⁃ In TREx293 cell lines, Tet ⁃ on system was used to induce the expression, which contained normal and abnormal atrophin ⁃ 1 full ⁃ length gene and GFP fusion proteins, respectively. Meanwhile, GFP ⁃ atrophin ⁃ 1 ⁃ Q19 and GFP ⁃ atrophin ⁃ 1 ⁃ Q66 recombinant plasmids were transiently transfected into 293T cells and SH⁃SY5Y cells respectively with LipofectamineTM 2000. The expression and localization of normal and abnormal atrophin ⁃ 1 fusion proteins in the cells were detected by light microscope with HE staining and fluorescence microscope; the ultrastructure of the cells were observed by electron microscope; the expression of GFP ⁃ atrophin ⁃ 1 fusion proteins were detected by Western blotting. Results In stable transfected system, the green fluorescence signals were detected in the nucleus, and abnormal proteins were not uniformly distributed while some were aggregated. Electron microscope showed shrinkaged nuclear membrane and abnormal intranuclear structure in abnormal cells. In transient transfection of 293T, Q66 protein aggregated in the nucleus which was detected as acidophilic bodies by HE staining⁃light microscope and atrophin⁃1 fusion protein as punctuate pattern by fluorescence microscope. Under the observation of electron microscope, great quantity of electron ⁃ dense substance accumulation, distinct shrinkage nuclear membrane and nucleus disappearance were seen in abnormal cells. The expression of GFP ⁃ atrophin ⁃ 1 fusion proteins were detected by Western blotting. The location of protein expression and Western blotting results in transient transfected SH⁃SY5Y cells system were essentially similar to those in transfected 293T cells. Conclusion In eukaryotic cell expression system, atrophin⁃1 with amplified polyglutamine chain may induce obvious aberrant aggregation in nucleus, and alteration of cellular appearance. The establishment of atrophin⁃1 expression eukaryotic cell model will provide the foundation for the research of pathogenesis of DRPLA, and therotic basis for further investigation of clincial intervention.

DOI：10.3969/j.issn.1672-6731.2011.01.016