title: Stable conditional expression and effect of C/ebpβ-LIP in adipocytes using the pSLIK system.
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.
authors.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
authors.forename: Cristina L
authors.initials: CL
authors.lastname: Esteves
authors.forename: Val
authors.initials: V
authors.lastname: Kelly
authors.forename: Valérie
authors.initials: V
authors.lastname: Bégay
authors.forename: Simon G
authors.initials: SG
authors.lastname: Lillico
authors.forename: Achim
authors.initials: A
authors.lastname: Leutz
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Seckl
authors.forename: Karen E
authors.initials: KE
authors.lastname: Chapman
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000235:genetics / Q000378:metabolism; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; C493411:Fabp4 protein, mouse; D050556:Fatty Acid-Binding Proteins; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D010729:Phosphoenolpyruvate Carboxykinase (GTP)
keywords: C/EBPβ-LIP; adipocyte; pSLIK; stable transfection