Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

C.B Sacramento,J.Z Moraes,P.M.A Denapolis,S.W Han

doi:10.1590/s0100-879x2010007500064

Abstract

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Highlights

Recent biotechnological advances have allowed the development of new technologies for the delivery of exogenous genetic material to mammalian cells
The well-known Simian virus 40 (SV40)-DNA-targeting sequences (DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors and the rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia were determined
To understand the influence of DTS on the transport of plasmids to the nucleus under hypoxia and normoxia, A293T cells were first transfected with plasmid vectors (Figure 1)

Summary

Introduction

Recent biotechnological advances have allowed the development of new technologies for the delivery of exogenous genetic material to mammalian cells. Functional genetic studies of these new technologies have resulted in a new therapeutic modality termed gene therapy. Of the several types of vectors available for gene therapy, naked DNA is used in 20% of current clinical trials This non-viral vector is easy to produce, handle, and purify and generates a low immune response, allowing repeated administrations if necessary. Its utility in gene therapy, is currently limited by its low rate of transfection. Gene expression levels can be increased by facilitating plasmid entry into the nucleus [1], since the main barrier for plasmid vector transfection is the nuclear envelope crossing [2,3,4,5]

Objectives

Methods

Results