Highly efficient regulation of gene expression by tetracycline in a replication-defective herpes simplex viral vector.
Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.
Laboratory of Tissue Repair and Gene Transfer, Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. fyao@rics.bwh.harvard.edu
Feng
F
Yao
Christoph
C
Theopold
Daniela
D
Hoeller
Oliver
O
Bleiziffer
Zheming
Z
Lu
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects / Q000821:virology; D011401:Promoter Regions, Genetic; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D018675:Toxicity Tests; D014709:Vero Cells / Q000187:drug effects / Q000821:virology; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*; D001616:beta-Galactosidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C083139:Tet O resistance protein, Bacteria; D014764:Viral Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; C064565:UL9 protein, Human herpesvirus 1; D004815:Epidermal Growth Factor; D020932:Nerve Growth Factor; D001616:beta-Galactosidase; D013752:Tetracycline