Construction of a new Hygromycin resistant Enhanced Green Fluorescent Protein (EGFP) Fusion vector (pEGFP-Hygro)

Green florescent Protein (GFP), originally isolated from the jellyfish Aequorea Victoria, has proven to be a useful reporter for monitoring gene expression and protein localization in vivo and in real time when fused to an intracellular or secretary protein. Unlike other bioluminescent reporters, GFP requires no additional proteins, substrates or co-factors to emit light. When irradiated with UV light or blue light, it emits green light, which enables the examination of expressed gene after expression. Enhanced green fluorescent protein (EGFP) is a red shifted variant of wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian systems. In order to construct a hygromycin resistant EGFP fusion vector we transferred the EGFP open reading frame of pEGFP-C1 (Clontech) to pCDNA3.1/Hygro/lacZ (Invitrogen) after excision of the lacZ fragment from the latter vector. Cloning confirmation procedures and EGFP expression of transfected MCF-7 breast cancer cell line, additionally with hygromycin resistance phenotype of the green shining cells following treatment of the transfected cells with hygromycin B revealed that our newly synthesized hygromycin resistant EGFP fusion shuttle vector was constructed successfully. This vector can be used as an easily detectable reporter and/or protein fusion vector for a number of basic experimental applications in situ and in vivo.

کلیدواژه ها:

fusion vector ، EGFP ، Hygromycin ، pEGFP-C1 ، pCDNA3.1/Hygro/lacZ