Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters.
BACKGROUND
The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX.


METHODS
We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX.


RESULTS
We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX.


CONCLUSIONS
Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.
Pluta
Krzysztof
K
Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center New Orleans, New Orleans, LA 70112, USA.
Luce
Milson J
MJ
Bao
Lili
L
Agha-Mohammadi
Siamak
S
Reiser
Jakob
J
D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D017690:Cell Transplantation; D002645:Chickens; D004318:Doxycycline / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D012516:Osteosarcoma / Q000473:pathology; D011401:Promoter Regions, Genetic; D012867:Skin / Q000166:cytology; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000235:genetics; D019076:Transgenes; D014183:Transplantation, Heterologous
D015534:Trans-Activators; D005914:Globins; D013752:Tetracycline; D004318:Doxycycline