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.
Yao
Feng
F
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
Theopold
Christoph
C
Hoeller
Daniela
D
Bleiziffer
Oliver
O
Lu
Zheming
Z
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