Evrogen has developed a novel mammalian expression vector that encodes KillerRed protein targeted to the cell membrane
KillerRed is a first genetically encoded photosensitiser allowing photoinactivation of proteins and precise cell killing in vivo.
Unlike chemical analogues, KillerRed can be directly expressed by target cells.
Upon green light irradiation KillerRed generates reactive oxygen species (ROS) that damage the neighbouring molecules.
To target KillerRed to cell membrane, it was fused with the membrane localisation signal (MLS) of neuromodulin.
Previously developed mitochondria-targeted KillerRed provided cell killing trough apoptotic pathway upon intensive light irradiation.
Irradiation of membrane-localised KillerRed leads to more effective and fast cell death, presumably due to lipid oxidation.
In addition, membrane targeted KillerRed has been shown to be suitable for the light induced cell killing within a developing zebrafish.
At the single cell stage zebrafish embryo was microinjected with a mixture of vectors driving expression of membrane-targeted KillerRed and green fluorescent protein under the control of CMV promoter.
Fluorescence was collected using standard FITC and TRITC filter sets.
A region containing a muscle cell expressing both proteins was irradiated with green light (40x objective, TRITC filter set, 10min) at 48 hours after fertilization.
By the end of 10min irradiation the cell already started changing its shape.
Within 20 minutes after irradiation was stopped the cell was disrupted completely.
In analogues experiments mitochondrially targeted KillerRed was shown to be of low efficiency.
