[Construction of the eukaryotic expression vector of MJD1 and its expression in SH-SY5Y cells].
OBJECTIVE
To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.


METHODS
The coding sequence of wild-type MJD1 and mutant MJD1 was amplified by PCR from pAS2-1-MJD20Q and pAS2-1-MJD68Q respectively. After being digested with BamH I and Hind III, the PCR products were inserted into pcDNA3. 1-Myc-His(-) B. The recombinant plasmids pcDNA3.1-Myc-His(-) B-MJD20Q and pcDNA3.1-Myc-His(-) B-MJD68Q were identified by enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into SH-SYSY cells and the expression of MJD1 in the transfected cells was analyzed by Western blot. The immunofluorescence of the transfected cells was examined using a confocal microscope to observe the formation of intranuclear aggregation.


RESULTS
Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into pcDNA3. 1-Myc-His(-) B. The expression of MJD1 in the transfected cells was confirmed by Western blot; The SH-SY5Y cells transfected with pcDNA3. 1-Myc-His(-) B-MJD68Q showed the formation of intranuclear aggregation, but the cells transfected with pcDNA3.1-Myc-His(-) B-MJD20Q did not show such phenomenon.


CONCLUSIONS
The eukaryotic expression vectors of MJD1 has been successfully constructed; The polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.
Tang
Jian-guang
JG
Department of Neurology, Xiangya Hospital, Central South University, Changsha 410008, China.
Tang
Bei-sha
BS
Shen
Lu
L
Jiang
Hong
H
Hu
Zhi-ping
ZP
Cao
Li
L
Xia
Kun
K
Cai
Fang
F
D000067696:Ataxin-1; D000067699:Ataxin-3; D000067528:Ataxins; D001483:Base Sequence; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D056892:Mediator Complex; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
C000593749:ATXN1 protein, human; D000067696:Ataxin-1; D000067528:Ataxins; C460107:MED12 protein, human; D056892:Mediator Complex; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D012097:Repressor Proteins; C092341:ATXN3 protein, human; D000067699:Ataxin-3