A novel inducible amplifier expression vector for high and controlled gene expression.
Safety and efficacy are both required for successful gene therapy. In this regard, our laboratory has created a novel expression system, pHi-Hot that combines inducible and amplifier strategies in one construct. In pHi-Hot, the first transcriptional unit contains an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. The magnitude of the amplified second gene expression can be regulated through manipulating the activity of the hsp promoter driving the Tat gene. Using the human interleukin-2 (IL-2) cytokine gene as the reporter gene, we demonstrated that moderate heat shock at 42 degrees C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were 35- to 70-fold higher than achieved by using the hsp promoter alone, and 10- to 35-fold higher than achieved by using the CMV promoter. Using inducible and amplifier strategies, we can achieve high and controlled gene expression levels from a single construct. Finally, we discuss the advantages of using these strategies in developing new targeting and inducible vectors for genetic research and gene therapy.
Luo
Phoebe
P
Gene Therapy Group, Department of Microbiology and Immunology, University of Arizona, Tucson, AZ 85721-0001, USA. phoebeluo2000@yahoo.com
He
Xianghui
X
Tsang
Tom C
TC
Harris
David T
DT
D000818:Animals; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured
D006360:Heat-Shock Proteins; D007376:Interleukin-2