Expression Cloning of Recombinant Escherichia coli lacZ Genes Encoding Cytoplasmic and Nuclear P-galactosidase Variants | ||
| Iranian Journal of Basic Medical Sciences | ||
| مقاله 9، دوره 14، شماره 4، مهر 2011، صفحه 369-375 اصل مقاله (376.3 K) | ||
| نوع مقاله: Original Article | ||
| شناسه دیجیتال (DOI): 10.22038/ijbms.2011.5026 | ||
| نویسندگان | ||
| Homayoun Naderian1؛ Zahra Rezvani1؛ Mohammad Ali Atlasi* 1؛ Hossein Nikzad1؛ Antoine AF de Vries2 | ||
| 1Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran | ||
| 2Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands | ||
| چکیده | ||
| Objective(s) Nonviral vector can be an attractive alternative to gene delivery in experimental study. In spite of some advantages in comparison with the viral vectors, there are still some limitations for efficiency of gene delivery in nonviral vectors. To determine the effective expression, the recombinant Escherichia coli lacZ genes were cloned into the different variants of pcDNA3.1 and then the mammalian cells were transfected. Methods and Materials The coding sequences of cytoplasmic and nuclear variants of lacZ gene were inserted downstream of the human cytomegalovirus immediate-early gene promoter of plasmid pcDNA3.1/myc-His C. The new cytoplasmic and nuclear constricts of E. coli p-galactosidase-coding sequences were introduced into HeLa cells with the aid of linear polyethylenimine and at 2 days post-transfection the cells were stained using 5- bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-gal). Results Restriction enzyme analyses revealed the proper insertion of E. coli p-galactosidase-coding sequences into the multiple cloning site of pcDNA3.1/myc-His C. The functionality of the resulting constructs designated pcDNA3.1-cyt.lacZ and pcDNA3.1-nls.lacZ(+) was confirmed by X-gal staining of HeLa cells transfected with these recombinant plasmids. While pcDNA3.1-cyt.lacZ directed the synthesis of cytoplasmically located p-galactosidase molecules, the p-galactosidase protein encoded by pcDNA3.1-nls.lacZ(+) was predominantly detected in the cell nucleus. Conclusion The expression of cytoplasmic and nuclear variant of LacZ gene confirmed the ability of pcDNA3.1 as versatility nonviral vector for the experimental gene delivery study in mammalian cells | ||
| کلیدواژهها | ||
| Expression cloning؛ Hela Cells؛ lacZ gene؛ pcDNA3.1/myc-His C؛ Polyethylenimine؛ Transfection | ||
| مراجع | ||
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