The development of an expression system for use in 3T3-L1 adipocytes

• Main Author: Hayward, A.
• Published: University of Cambridge 1997
• Subjects: 612.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603897

3T3-L1 cells may be propagated as fibroblasts, but when grown to confluency may be induced to terminally differentiate into insulin responsive adipocytes which accumulate high levels of fat and express LUT4. The strength of different promoters in both 3T3-L1 fibroblasts and adipocytes was investigated. It was found that the RSV promoter functioned well in fibroblasts but poorly in adipocytes, the CMV and EF-1α (elongation factor-1α) promoters both maintained high level expression in both cell stages and the aP2 was adipocyte specific. Initial attempts to generate an inducible adipocyte specific expression system focused on altering the LacSwitch (Stratagene) inducible mammalian expression system for use in adipocytes, although these studies proved unsuccessful. The aP2 promoter was therefore used to control expression of exogenous genes until four days after the initiation of differentiation. Initially the expression system was characterised with the CAT reporter gene before several proteins involved in insulin signal transduction (a dominant negative interfering mutant of the p85 subunit of PI3-K (Δp85), Wt and N17/V12 Rac, WT and C459S SHPTP2) were expressed. The time course of gene expression during differentiation and the effects of expression of insulin signalling to glucose transport and p70S6 kinase activation were monitored, but few significant effects were found. Finally, studies wre made into the time course of glucose transporter expression and GLUT isoform distribution during 3T3-L1 cell differentiation. The aP2 promoter has been successfully used to drive adipocyte specific expression of proteins in 3T3-L1 adipocytes, but a further element of inducible control would be advantageous to prevent expression of exogenous proteins until after completion of differentiation.