Cloning and characterization of ovine alphaS1-casein gene promoter: a transfection study in rat mammary gland cell line.
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 alphaS1-CSNGP. For the first time we have cloned and sequenced region extending from - 2136 to +49 bp containing 5'-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/VspI showed highest fluorescence followed by pGFP-P, pGFP-F/SpeI, pGFPminimal and pGFP-D.
Bhure
Sanjeevkumar
S
Project Directorate on Animal Disease Monitoring Surveillance (PD_ADMAS). Hebbal, Bangalore, Karnataka, 560 024. India. sdbhure@rediffmail.com
Sharma
Bhaskar
B
D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D012756:Sheep; D014162:Transfection
D002364:Caseins; D017931:DNA Primers; D049452:Green Fluorescent Proteins