A versatile tool for conditional gene expression and knockdown

Abstract

Drug-inducible systems allowing the control of gene expression in mammalian cells are invaluable tools for genetic research, and could also fulfill essential roles in gene- and cell-based therapy. Currently available systems, however, often have limited in vivo functionality because of leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. Here we describe a lentiviral vector-based, conditional gene expression system for drug-controllable expression of polymerase (Pol) II promoter-driven transgenes or Pol III promoter-controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). This system has great robustness and versatility, governing tightly controlled gene expression in cell lines, in embryonic or hematopoietic stem cells, in human tumors xenotransplanted into nude mice, in the brain of rats injected intraparenchymally with the vector, and in transgenic mice generated by infection of fertilized oocytes. These results open up promising perspectives for basic or translational research and for the development of gene-based therapeutics.

Tightly regulated gene expression in human hematopoietic stem cells after transduction with helper-dependent Ad5/35 vectors

“…The first vector (HD-Ad5/35.Tet-1) contained a fusion between the Krüppel-associated box (KRAB) domain and the tetracycline repressor (TetR). In this system, tTR-KRAB-mediated repression of a PGK promoter, juxtaposed to Tet operator sequences can be reversibly controlled by Dox [22]. The second vector (HD-Ad5/35.Tet-2) contained the standard Tet-on expression system, using an Tet transactivator (tTA) for regulated transgene expression [23].…”

“…Binding of DNA binding proteins fused to KRAB results in histone deacetylation and methylation, thus creating a local heterochromatin state and inactivation of promoters that are 2 to 3 kb up-or downstream of the binding site [29]. It has been shown that tTR-KRAB mediated repression of cellular Pol-II and Pol-III promoters juxtaposed to the TetO can be reversibly controlled by Dox [22]. These studies were done with transgenes cassettes integrated into the cell genome.…”

“…For packaging the Gigapack III Plus Packaging Extract Kit (Stratagene, La Jolla, CA) was used. pHD-Ad.Tet-1 contains the Tetinducible GFP expression cassette from pLVPT-tTRKRAB [22]. We first constructed pBStTRKRAB.…”

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

“…Next, the same construct can be used to transduce hematopoietic stem cells isolated from the C3H.TgN (SRA-rtTA) transgenic mice and generate bone marrow chimeras that would express a gene-of-interest in mature macrophages and, possibly, myeloid dendritic cells, after stimulation with doxycycline. Finally, the same lentiviral vector may be used to generate transgenic mice on the C3H.TgN (SRA-rtTA) genetic background (Pfeifer et al, 2002) (Szulc et al, 2006) that would express the gene-of-interest in macrophages after induction with doxycycline. In the future, it may be used in combination with novel systems for tetracycline inducible expression of transgene and/or RNAi (Szulc et al, 2006) and macrophage progenitor expansion (Odegaard et al, 2006;Wang et al, 2006) to make genetic and functional analysis of macrophages more efficient.…”

A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation

“…To enable the external control over the activity of the device, an inducible OFF-switch, implemented as the rtTR-KRAB repressor, 24 was introduced. This switch reset the device by external chemical signal and shut down production of the anti-inflammatory effectors when they were no longer required.…”