Regulating gene expression using self-inactivating lentiviral vectors containing the mifepristone-inducible system

Olga Sirin, Frank Park

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Methods to regulate gene expression in vitro and in vivo are currently areas of intense research. The present study, therefore, was designed to determine the efficacy of transgene expression using the GeneSwitch mifepristone-regulatable system within the context of an integrating HIV-1 vector. Lentiviral transfer plasmids expressing the red (DsRed2) and green fluorescent protein (EGFP) markers were constructed for in vitro assessment on the basal and mifepristone-induced cell activation levels by FACS analyses. In our design, efficient cell activation and transgene expression were found using a binary lentivector system i.e., the trans-activator, Switch, and the inducible promoter-transgene expression cassette were cloned into separate vectors. Note that the Switch trans-activator performed optimally when cloned into the reverse-orientation, but the inducible promoter containing lentivector did not appear to be dependent upon the orientation within the lentivector backbone. This binary lentivector system resulted in tightly regulated transgene expression, with low basal cell activation in the absence of mifepristone (MFP). Upon induction, a 41- to 275-fold increase in the number of DsRed2- and EGFP-positive cells were detected (n=3). To determine the inducing ability of the GeneSwitch, we cloned the human α1-antitrypsin cDNA into the optimal lentiviral vector and transduced HeLa and Huh7 cells at increasing lentivector doses as determined by p24 Gag ELISA. We found that MFP could induce the expression of hAAT protein in HeLa cells from 310 to 15,000 ng hAAT/106 cells/24 h, which was a 48-fold induction. Similar results were observed in huH7 cells. In all, this study demonstrates that the GeneSwitch system can be designed within the context of a lentiviral vector for in vitro gene transfer, and this may also provide a viable method for temporally regulating gene expression for therapeutic applications in vivo or ex vivo.

Original languageEnglish (US)
Pages (from-to)67-77
Number of pages11
JournalGene
Volume323
Issue number1-2
DOIs
StatePublished - Dec 24 2003

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Mifepristone
Transgenes
Gene Expression
Trans-Activators
Green Fluorescent Proteins
HeLa Cells
HIV-1
Plasmids
Complementary DNA
Enzyme-Linked Immunosorbent Assay
Research
Genes
In Vitro Techniques
Proteins

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Regulating gene expression using self-inactivating lentiviral vectors containing the mifepristone-inducible system. / Sirin, Olga; Park, Frank.

In: Gene, Vol. 323, No. 1-2, 24.12.2003, p. 67-77.

Research output: Contribution to journalArticle

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