Cloning and characterization ovine áS1- casein gene promoter: A transfection study in rat mammary gland cell line

Abstract

Promoter regions of milk protein genes are frequently used to produce pharmaceutically and medically important proteins in the mammary gland of transgenic animals and also can be used for the construction of an inducible eukaryotic expression vector. The aim of the present study was to clone, sequence and characterize the regulatory elements in ovine aS1-CSNGP. For the first time we have cloned and sequenced region extending from 22136 to þ49 bp containing 50-flanking region and exon I. Computational analysis of the sequence showed presence of core promoter elements viz., TATA box, CAAT box and initiator sequence. Mammary gland specific sequences included MGF/STAT 5, MPBF, Yu Lee 2, 4 and 5, Oka box C and hormone responsive elements (HRE) viz., GRE, PRE, PRL, IRE and also Polyoma enhancer 3 sequences. Computational analysis data is validated by following the reporter gene expression studies in rat breast cell line. Six reporter gene constructs under the control of full length, proximal, distal, minimal and proximal–distal fused promoter segments were constructed to assess the effect of presence or absence of few selected regulatory elements on expression ability of the promoter. Based on qualitative evaluation of fluorescence, the pGFP-F/Vsp I showed highest fluorescence followed by pGFP-P, pGFP-F/Spe I, pGFPminimal and pGFP-D.