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Stable conditional expression and effect of C/ebpβ-LIP in adipocytes using the pSLIK system.

Esteves CL, Kelly V, Bégay V, Lillico SG, Leutz A, Seckl JR, Chapman KE - J. Mol. Endocrinol. (2013)

Bottom Line: Increased C/EBPβ-LIP in mature adipocytes down-regulated C/ebpβ target genes including 11β-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4 but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/ebpβ-LIP in 3T3-L1 adipocytes is not a general effect.Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/ebpβ-LIP levels.The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology Unit, Queen's Medical Research Institute, University/BHF Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, UK. cristina.esteves@ed.ac.uk

ABSTRACT
Murine 3T3-L1 adipocytes are widely used as a cellular model of obesity. However, whereas transfection of 3T3-L1 preadipocytes is straightforward, ectopic gene expression in mature 3T3-L1 adipocytes has proved challenging. Here, we used the pSLIK vector system to generate stable doxycycline-inducible expression of the liver-enriched inhibitor protein isoform of CCAAT/enhancer binding protein β (C/ebpβ (Cebpb)) (C/EBPβ-LIP) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/ebpβ-LIP impairs adipocyte differentiation, the C/ebpβ-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBPβ-LIP in mature adipocytes down-regulated C/ebpβ target genes including 11β-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4 but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/ebpβ-LIP in 3T3-L1 adipocytes is not a general effect. Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/ebpβ-LIP levels. The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology.

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The pSLIK-C/EBPβ-LIP construct and generation of stably transfected 3T3-L1 preadipocytes. (A) Schematic representation of the pSLIK Tet-On empty vector (top; vector, pSLIK) and construct used to generate stable 3T3-L1 preadipocytes conditionally overexpressing C/EBPβ-LIP (bottom; LIP, pSLIK-C/EBPβ-LIP). C/EBPβ-LIP and GFP are co-expressed from the same transcript. The constitutively active ubiquitin c (Ubi-c) promoter drives expression of the Tet activator (rtTA3) and neomycin resistance gene (Neo; for selection). Addition of DOX causes binding of rtTA3 to the TRE/CMV promoter (TRE), inducing expression of GFP or C/EBPβ-LIP-IRES-GFP. (B) FACS analysis and sorting of 3T3-L1 preadipocytes (5×106 cells) stably transfected with pSLIK-C/EBPβ-LIP (LIP) or pSLIK vector (vector) following DOX treatment (1 μg/ml, 24 h) to induce GFP. Upper panels, forward scatter (FSC) vs side scatter (SSC) dot plots, showing the populations of single cells (circled) that were analysed and sorted for GFP expression (FSC vs GFP intensity plots; middle row of panels) and histogram showing the number of cells for GFP intensity (lower panels). Middle and lower panels show the enrichment of GFP-positive cells for both pSLIK-C/EBPβ-LIP and pSLIK cells after sorting (right panels) resulting from the removal of low or non-GFP-expressing preadipocytes (arrow; left panels).
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fig1: The pSLIK-C/EBPβ-LIP construct and generation of stably transfected 3T3-L1 preadipocytes. (A) Schematic representation of the pSLIK Tet-On empty vector (top; vector, pSLIK) and construct used to generate stable 3T3-L1 preadipocytes conditionally overexpressing C/EBPβ-LIP (bottom; LIP, pSLIK-C/EBPβ-LIP). C/EBPβ-LIP and GFP are co-expressed from the same transcript. The constitutively active ubiquitin c (Ubi-c) promoter drives expression of the Tet activator (rtTA3) and neomycin resistance gene (Neo; for selection). Addition of DOX causes binding of rtTA3 to the TRE/CMV promoter (TRE), inducing expression of GFP or C/EBPβ-LIP-IRES-GFP. (B) FACS analysis and sorting of 3T3-L1 preadipocytes (5×106 cells) stably transfected with pSLIK-C/EBPβ-LIP (LIP) or pSLIK vector (vector) following DOX treatment (1 μg/ml, 24 h) to induce GFP. Upper panels, forward scatter (FSC) vs side scatter (SSC) dot plots, showing the populations of single cells (circled) that were analysed and sorted for GFP expression (FSC vs GFP intensity plots; middle row of panels) and histogram showing the number of cells for GFP intensity (lower panels). Middle and lower panels show the enrichment of GFP-positive cells for both pSLIK-C/EBPβ-LIP and pSLIK cells after sorting (right panels) resulting from the removal of low or non-GFP-expressing preadipocytes (arrow; left panels).

Mentions: To generate a vector to conditionally express C/EBPβ-LIP, the encoding cDNA was cloned and recombined in the MBA-266 and the pSLIK-Neo MBA-235 vectors, respectively, producing the pSLIK-C/EBPβ-LIP vector in which a tetracycline response element drives expression of C/EBPβ-LIP from a minimal CMV promoter in the presence of DOX (a Tet-ON system) and a contiguous IRES allows for GFP expression. Constitutive expression of the reverse Tet transactivator (rtTA3) and the neomycin resistance gene (Neo) is driven by the ubiquitin-c (Ubi-c) promoter (Fig. 1A). In order to express C/EBPβ-LIP in mature adipocytes, we first generated stably transfected 3T3-L1 preadipocytes that, in contrast to fully differentiated adipocytes, could be readily transfected with pSLIK-C/EBPβ-LIP or the ‘empty’ vector. This was highly efficient allowing selection of transfectants with geneticin as a total pool of cells, avoiding clonal differences due to plasmid integration site. To ensure that only transfected cells were used in the subsequent experiments, preadipocytes were sorted by flow cytometry for GFP expression 24 h after incubation with DOX, with yields of 26 and 56% for pSLIK-C/EBPβ-LIP and pSLIK-transfectants respectively (Fig. 1B). In the absence of DOX, only 0.9% of pSLIK-C/EBPβ-LIP and 4.3% of pSLIK preadipocytes were GFP positive, demonstrating low spontaneous expression in the transfected preadipocytes. pSLIK-C/EBPβ-LIP preadipocytes were then differentiated into mature adipocytes in DOX-free medium (to avoid the inhibitory effects of C/EBPβ-LIP upon adipocyte differentiation) and subsequent DOX treatment robustly increased cellular C/EBPβ-LIP levels in the mature adipocytes (Fig. 2A and B). Following removal of DOX from the cell medium, C/EBPβ-LIP decreased as expected, showing that it can be transiently induced (Fig. 2B). Importantly, in the absence of DOX, pSLIK-C/EBPβ-LIP adipocytes showed comparable low endogenous levels of C/EBPβ-LIP to control adipocytes harbouring the pSLIK empty vector (Fig. 2A).


Stable conditional expression and effect of C/ebpβ-LIP in adipocytes using the pSLIK system.

Esteves CL, Kelly V, Bégay V, Lillico SG, Leutz A, Seckl JR, Chapman KE - J. Mol. Endocrinol. (2013)

The pSLIK-C/EBPβ-LIP construct and generation of stably transfected 3T3-L1 preadipocytes. (A) Schematic representation of the pSLIK Tet-On empty vector (top; vector, pSLIK) and construct used to generate stable 3T3-L1 preadipocytes conditionally overexpressing C/EBPβ-LIP (bottom; LIP, pSLIK-C/EBPβ-LIP). C/EBPβ-LIP and GFP are co-expressed from the same transcript. The constitutively active ubiquitin c (Ubi-c) promoter drives expression of the Tet activator (rtTA3) and neomycin resistance gene (Neo; for selection). Addition of DOX causes binding of rtTA3 to the TRE/CMV promoter (TRE), inducing expression of GFP or C/EBPβ-LIP-IRES-GFP. (B) FACS analysis and sorting of 3T3-L1 preadipocytes (5×106 cells) stably transfected with pSLIK-C/EBPβ-LIP (LIP) or pSLIK vector (vector) following DOX treatment (1 μg/ml, 24 h) to induce GFP. Upper panels, forward scatter (FSC) vs side scatter (SSC) dot plots, showing the populations of single cells (circled) that were analysed and sorted for GFP expression (FSC vs GFP intensity plots; middle row of panels) and histogram showing the number of cells for GFP intensity (lower panels). Middle and lower panels show the enrichment of GFP-positive cells for both pSLIK-C/EBPβ-LIP and pSLIK cells after sorting (right panels) resulting from the removal of low or non-GFP-expressing preadipocytes (arrow; left panels).
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fig1: The pSLIK-C/EBPβ-LIP construct and generation of stably transfected 3T3-L1 preadipocytes. (A) Schematic representation of the pSLIK Tet-On empty vector (top; vector, pSLIK) and construct used to generate stable 3T3-L1 preadipocytes conditionally overexpressing C/EBPβ-LIP (bottom; LIP, pSLIK-C/EBPβ-LIP). C/EBPβ-LIP and GFP are co-expressed from the same transcript. The constitutively active ubiquitin c (Ubi-c) promoter drives expression of the Tet activator (rtTA3) and neomycin resistance gene (Neo; for selection). Addition of DOX causes binding of rtTA3 to the TRE/CMV promoter (TRE), inducing expression of GFP or C/EBPβ-LIP-IRES-GFP. (B) FACS analysis and sorting of 3T3-L1 preadipocytes (5×106 cells) stably transfected with pSLIK-C/EBPβ-LIP (LIP) or pSLIK vector (vector) following DOX treatment (1 μg/ml, 24 h) to induce GFP. Upper panels, forward scatter (FSC) vs side scatter (SSC) dot plots, showing the populations of single cells (circled) that were analysed and sorted for GFP expression (FSC vs GFP intensity plots; middle row of panels) and histogram showing the number of cells for GFP intensity (lower panels). Middle and lower panels show the enrichment of GFP-positive cells for both pSLIK-C/EBPβ-LIP and pSLIK cells after sorting (right panels) resulting from the removal of low or non-GFP-expressing preadipocytes (arrow; left panels).
Mentions: To generate a vector to conditionally express C/EBPβ-LIP, the encoding cDNA was cloned and recombined in the MBA-266 and the pSLIK-Neo MBA-235 vectors, respectively, producing the pSLIK-C/EBPβ-LIP vector in which a tetracycline response element drives expression of C/EBPβ-LIP from a minimal CMV promoter in the presence of DOX (a Tet-ON system) and a contiguous IRES allows for GFP expression. Constitutive expression of the reverse Tet transactivator (rtTA3) and the neomycin resistance gene (Neo) is driven by the ubiquitin-c (Ubi-c) promoter (Fig. 1A). In order to express C/EBPβ-LIP in mature adipocytes, we first generated stably transfected 3T3-L1 preadipocytes that, in contrast to fully differentiated adipocytes, could be readily transfected with pSLIK-C/EBPβ-LIP or the ‘empty’ vector. This was highly efficient allowing selection of transfectants with geneticin as a total pool of cells, avoiding clonal differences due to plasmid integration site. To ensure that only transfected cells were used in the subsequent experiments, preadipocytes were sorted by flow cytometry for GFP expression 24 h after incubation with DOX, with yields of 26 and 56% for pSLIK-C/EBPβ-LIP and pSLIK-transfectants respectively (Fig. 1B). In the absence of DOX, only 0.9% of pSLIK-C/EBPβ-LIP and 4.3% of pSLIK preadipocytes were GFP positive, demonstrating low spontaneous expression in the transfected preadipocytes. pSLIK-C/EBPβ-LIP preadipocytes were then differentiated into mature adipocytes in DOX-free medium (to avoid the inhibitory effects of C/EBPβ-LIP upon adipocyte differentiation) and subsequent DOX treatment robustly increased cellular C/EBPβ-LIP levels in the mature adipocytes (Fig. 2A and B). Following removal of DOX from the cell medium, C/EBPβ-LIP decreased as expected, showing that it can be transiently induced (Fig. 2B). Importantly, in the absence of DOX, pSLIK-C/EBPβ-LIP adipocytes showed comparable low endogenous levels of C/EBPβ-LIP to control adipocytes harbouring the pSLIK empty vector (Fig. 2A).

Bottom Line: Increased C/EBPβ-LIP in mature adipocytes down-regulated C/ebpβ target genes including 11β-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4 but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/ebpβ-LIP in 3T3-L1 adipocytes is not a general effect.Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/ebpβ-LIP levels.The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology Unit, Queen's Medical Research Institute, University/BHF Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, UK. cristina.esteves@ed.ac.uk

ABSTRACT
Murine 3T3-L1 adipocytes are widely used as a cellular model of obesity. However, whereas transfection of 3T3-L1 preadipocytes is straightforward, ectopic gene expression in mature 3T3-L1 adipocytes has proved challenging. Here, we used the pSLIK vector system to generate stable doxycycline-inducible expression of the liver-enriched inhibitor protein isoform of CCAAT/enhancer binding protein β (C/ebpβ (Cebpb)) (C/EBPβ-LIP) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/ebpβ-LIP impairs adipocyte differentiation, the C/ebpβ-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBPβ-LIP in mature adipocytes down-regulated C/ebpβ target genes including 11β-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4 but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/ebpβ-LIP in 3T3-L1 adipocytes is not a general effect. Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/ebpβ-LIP levels. The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology.

Show MeSH
Related in: MedlinePlus