Expression Cloning of Recombinant Escherichia coli lacZ Genes Encoding Cytoplasmic and Nuclear P-galactosidase Variants

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 pcDNA۳.۱ 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 pcDNA۳.۱/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 ۲ days post-transfection the cells were stained using ۵- bromo-۴-chloro-۳-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 pcDNA۳.۱/myc-His C. The functionality of the resulting constructs designated pcDNA۳.۱-cyt.lacZ and pcDNA۳.۱-nls.lacZ(+) was confirmed by X-gal staining of HeLa cells transfected with these recombinant plasmids. While pcDNA۳.۱-cyt.lacZ directed the synthesis of cytoplasmically located p-galactosidase molecules, the p-galactosidase protein encoded by pcDNA۳.۱-nls.lacZ(+) was predominantly detected in the cell nucleus. Conclusion The expression of cytoplasmic and nuclear variant of LacZ gene confirmed the ability of pcDNA۳.۱ as versatility nonviral vector for the experimental gene delivery study in mammalian cells

Keywords:

Expression cloning , Hela Cells , lacZ gene , pcDNA۳.۱/myc-His C , Polyethylenimine , Transfection