To generate an expression plasmid for optimal in vivo imaging in mice, a modified variant of the Rhodopseudomonas palustris bacteriophytochrome RpBphP2, called iRFP for infraRed Fluorescent Protein (excitation/emission maxima at 690/713 nm) 31 (link), was cloned from the pShuttle-CMV-iRFP plasmid (#31856, Addgene, Cambridge, MA, U.S.A.). A BglII/NotI restriction double digest was used to insert the iRFP gene into the pEGFP-N1 plasmid (Clontech, Mountain View, CA, U.S.A.) in place of the eGFP gene. This new plasmid allowed for constitutive expression of iRFP directed by the CMV promoter.
The iRFP plasmid was transformed into Escherichia coli bacterial strain TOP10 and pure, supercoiled plasmid for transfection was isolated with the EndoFree Plasmid Maxi kit (#12363, Qiagen, Hilden, Germany) using manufacturer’s instructions. The identity of the plasmid preparation was confirmed by DNA sequencing followed by measurement of the purity and concentration through the 260/280 nm ratio of UV absorbance using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, U.S.A.).
The iRFP plasmid was transformed into Escherichia coli bacterial strain TOP10 and pure, supercoiled plasmid for transfection was isolated with the EndoFree Plasmid Maxi kit (#12363, Qiagen, Hilden, Germany) using manufacturer’s instructions. The identity of the plasmid preparation was confirmed by DNA sequencing followed by measurement of the purity and concentration through the 260/280 nm ratio of UV absorbance using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, U.S.A.).