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Delivery of b-ZIP inhibitor protein into 3T3-L1 cells to study time-bound preadipocyte differentiation process

Nishtha Sharma

A thorough study of transcription factors involved in preadipocyte differentiation would allow for manipulation of adipocyte physiology, cell number thus will help in controlling obesity and related morbidities. Basic leucine zippers (b-ZIP) are eukaryotic class of transcription factors (TFs) having pivotal roles in various processes including regulation of cellular growth and differentiation. CCAAT-enhancer binding proteins (C/EBPs) are a family of b-ZIP TFs that regulate adipocyte differentiation. Hormonal induction of 3T3-L1 preadipocytes leads to expression of CCAAT-enhancer binding protein C/EBP β and C/EBP δ. These transcription factors further induces the expression of C/EBP α, which along with PPARγ, activates all adipocyte-specific genes. A designed dominant negative A-C/EBP which possesses a leucine zipper but lacks functional DNA-binding and transactivation domains, forms stable inactive heterodimers with C/EBPs in vitro and inhibits differentiation of 3T3 cells. In this study, delivery of expressed and purified A-C/EBP protein with Lipofectamine 2000 for few hours results in lesser lipid accumulation in differentiated cells in comparison to the cells transfected with A-C/EBP gene. Gene expression analysis of adipocyte-specfic genes and western blotting of target proteins have proven the degradation of target genes within few hours of protein transfection. ChIP PCR has provided the evidences of lesser binding of C/EBPβ to promoter region of adipocyte-marker genes in transfected cells