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Variability of inducible expression across the hematopoietic system of tetracycline transactivator transgenic mice.

ABSTRACT: The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.

SUBMITTER: Takiguchi M

PROVIDER: S-EPMC3543435 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Variability of inducible expression across the hematopoietic system of tetracycline transactivator transgenic mice.

Takiguchi Megumi M Dow Lukas E LE Prier Julia E JE Carmichael Catherine L CL Kile Benjamin T BT Turner Stephen J SJ Lowe Scott W SW Huang David C S DC Dickins Ross A RA

PloS one 20130111 1

The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell ty ...[more]

PMID: 23326559

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Project description:IntroductionThe controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones.MethodsWe developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells. This platform has three basic constructs: regulator, responder, and destination vectors. The modular platform is compatible with both the TetOff (tTA) and TetOn (rtTA) systems. The modular Gateway recombineering-compatible components facilitate rapidly generating vectors to genetically modify mammalian cells. We apply this system to use the elongation factor 1? (EF1?) promoter to drive doxycycline-regulated expression of both the fluorescent marker mCherry and an engineered Gs-coupled GPCR "Rs1" separated by a 2A ribosomal skip site.ResultsWe show that our combined expression construct drives expression of both the mCherry and Rs1 transgenes in a doxycycline-dependent manner. We successfully target the expression construct into the Rosa26 locus of mouse embryonic stem (ES) cells. Rs1 expression in mouse ES cells increases cAMP accumulation via both basal and ligand-induced Gs mechanisms and is associated with increased embryoid body size. Heterozygous mice carrying the Rs1 expression construct showed normal growth and weight, and developed small increases in bone formation that could be observed in the calvaria.ConclusionsOur results demonstrate the feasibility of a single-vector strategy that combines both the tTA and TRE tetracycline-regulated components for use in cells and mouse models. Although the EF1? promoter is useful for driving expression in pluripotent cells, a single copy of the EF1? promoter did not drive high levels of mCherry and Rs1 expression in the differentiated tissues of adult mice. These findings indicate that promoter selection is an important factor when developing transgene expression models.

Dataset Information

Variability of inducible expression across the hematopoietic system of tetracycline transactivator transgenic mice.

Publications

Variability of inducible expression across the hematopoietic system of tetracycline transactivator transgenic mice.

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