Biopolymers and cell. 2007; 23 (2): 100 - 107

Plasmid vector construction for the human preproinsulin gene delivery into mammalian cells

O. K. Toporova, S. D.Kyrylenko, D. M. Irodov, V. A. Kordium

Eukaryotic expression vector for human preproinsulin gene has been constructed for further delivery into non-endocrine mammalian cells in vitro and in situ for the development of experimental gene therapy for Type 1 diabetes. Vector construction consists of two independent modules, namely, bacterial plasmid sequence that allows its replication in Escherichia coli cells, and expression cassette for the target and marker genes which is flanked by inverted terminal repeats of human adeno-associated virus. To ensure the constitutive expression, human preproinsulin gene was subcloned under the control of the strong ubiquitous immediate early gene promoter/enhancer of human cytomegalovirus in cassette. The hepatitis B viral enhancer 1 was subcloned into cassette to increase the target transgene expression. After vector construction in vitro transfer into mammalian cells of different origin, the stable transformants were obtained and the availability of target human preproinsulin transgene in the transformed cells has been confirmed by PCR analysis.

Keywords: human preproinsulin gene, expression vector, stable transformants.