Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia.
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.
Sacramento
C B
CB
Centro Interdisciplinar de Terapia Gênica, Universidade Federal de São Paulo.
Moraes
J Z
JZ
Denapolis
P M A
PM
Han
S W
SW
D000818:Animals; D001483:Base Sequence / Q000235:genetics*; D015687:Cell Hypoxia / Q000235:genetics*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D013539:Simian virus 40 / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D042461:Vascular Endothelial Growth Factor A