Hematopoietic cytokine-inducible gene expression from retroviral vectors.
Retroviral vectors capable of cytokine-inducible gene expression will be useful for a number of gene therapy applications. We explored one mechanism whereby cytokine inducibility may be imparted to the retroviral U3 promoter/enhancer by utilizing the JAK-STAT signal transduction pathway that is activated by a number of hematopoietic cytokines. We used PCR mutagenesis to insert a consensus binding site for the ubiquitous transcription factor Sp1 into the Moloney murine leukemia virus U3 followed by the insertion of multimers of a STAT-binding oligonucleotide with the core sequence 5'-TTCCCGGAA. After insertion of the modified U3s into a retroviral vector expressing the luciferase reporter gene and transduction of the HepG2 cell line, luciferase expression was induced with recombinant human IFN-gamma. The level of induction reached a maximum of 9.9-fold higher than the uninduced vector when the Sp1-U3 contained four STAT oligos. When this optimal vector was compared with the wild-type and Sp1 vectors, respective values of 17.9- and 16.7-fold higher expression were achieved with IFN-gamma treatment. Retroviral vectors incorporating these cytokine-inducible U3s will be useful for gene therapy in a number of situations involving gene transfer to hematopoietic, hepatic and other cytokine-responsive cell types.
Saylors
R L
RL
Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock 72202, USA.
Stine
K C
KC
Derrick
J
J
D002460:Cell Line; D016207:Cytokines / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000235:genetics*; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection / Q000379:methods*
D016207:Cytokines; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D007371:Interferon-gamma; D008156:Luciferases