Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector

Citation
R. Pollock et al., Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector, P NAS US, 97(24), 2000, pp. 13221-13226
Citations number
23
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
00278424 → ACNP
Volume
97
Issue
24
Year of publication
2000
Pages
13221 - 13226
Database
ISI
SICI code
0027-8424(20001121)97:24<13221:DOASDG>2.0.ZU;2-Z
Abstract
Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robu st, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural produ ct dimerizer rapamycin was optimized for retroviral delivery by fusing a hi ghly potent chimeric activation domain to the rapamycin-binding domain of F KBP-rapamycin-associated protein (FRAP). This improvement led to an increas e in both the potency and maximal levels of gene expression induced by rapa mycin, or nonimmunosuppressive rapamycin analogs. The modified transcriptio n factor cassette was incorporated along with a target gene into a single r apamycin-responsive retrovirus. Cell pools stably transduced with the singl e virus system displayed negligible basal expression and gave induction rat ios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression w ere comparable to those obtained with the constitutive retroviral long term inal repeat and the single virus system performed well in four different ma mmalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible ex pression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus shoul d facilitate its use in the study of gene function in a broad range of cell types.