Journal Article A Comparative Study of The Internal Ribosome Entry Sitecontaining Expression System

Murakami, Manabu  ,  Murakami, Agnieszka A  ,  Ohba, Takayoshi

68 ( 1 )  , pp.44 - 51 , 2017-10-05 , 弘前大学大学院医学研究科・弘前医学会
We analyzed transient and stable eukaryotic gene expression using internal ribosome entry site (IRES)and green fluorescent protein (GFP)-S65T (IRES-GFP) expression systems. The IRES-containing vector enablessimple construction of a plasmid and easy detection of transfected cells, as well as an estimation of the levels of thegene of interest. This system is useful for measuring changes in the intracellular calcium concentration using thefluorescent calcium indicator Fura-2 AM, although subcellular localization of the protein of interest is not detectable.Importantly, the IRES-GFP plasmid showed delayed expression and an approximate 40% reduction in fluorescenceintensity, which suggests that usage of IRES-containing expression system might cause under-estimation of desiredgene. Although this technique has some disadvantages, the IRES-containing vector is simple and straightforward for gene expression analysis, comparing with co-expression procedure of multiple plasmids.

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