In vivo image analysis using iRFP transgenic mice

Abstract

Fluorescent proteins with light wavelengths within the optical window are one of the improvements in in vivo imaging techniques. Near-infrared (NIR) fluorescent protein (iRFP) is a stable, nontoxic protein that emits fluorescence within the NIR optical window without the addition of exogenous substrate. However, studies utilizing an in vivo iRFP model have not yet been published. Here, we report the generation of transgenic iRFP mice with ubiquitous NIR fluorescence expression. iRFP expression was observed in approximately 50% of the offspring from a matings between iRFP transgenic and WT mice. The serum and blood cell indices and body weights of iRFP mice were similar to those of WT mice. Red fluorescence with an excitation wavelength of 690 nm and an emission wavelength of 713 nm was detected in both newborn and adult iRFP mice. We also detected fluorescence emission in whole organs of the iRFP mice, including the brain, heart, liver, kidney, spleen, lung, pancreas, bone, testis, thymus, and adipose tissue. Therefore, iRFP transgenic mice may therefore be a useful tool for various types of in vivo imaging.

Fig. 1.

Generation of transgenic iRFP mice. A: iRFP cDNA was fused to the CAG promoter. The arrows indicate the genotyping primers. B-F: iRFP mice were easily identified using an IVIS Spectrum system equipped with 710 nm excitation and 780 nm emission filters. B: Four lines of iRFP transgenic F0 mice and iRFP-negative WT. C: Newborn iRFP transgenic mice from line 846 (marked with an asterisk) and a WT littermate. D: Newborn iRFP transgenic mice from line 867 (marked with an asterisk) and a WT littermate. E: Whole-body imaging of WT and iRFP mice before autopsy. F: Imaging of whole organs from WT and iRFP mice.

Fig. 2.

iRFP is expressed in individual organs. A-L: Images under near-infrared light. A’-L’: Images under visible light. Left, WT; middle, line 846; right, line 867.

Fig. 3.

Expression of iRFP in hematopoietic cells. A: Expression of iRFP in WT and iRFP hematopoietic cells after lysing red blood cells. B: FACS analysis of various blood cell markers (B220: B cell; Gr1: granulocyte; CD3: T cell; Mac1: monocyte; Ter119: erythrocyte). All blood cell types analyzed expressed iRFP. C: The expression of various blood cell markers in hematopoietic cells from WT and iRFP mice was similar. The data indicate as the means ± SEM. WT, n=4; lines 846 and 867, n=3.

Fig. 4.

Expression of iRFP in hematopoietic cells from BM cells-transplanted mice. A: Expression of iRFP in WT and iRFP hematopoietic cells after lysing red blood cells. B: FACS analysis of various blood cell markers (B220: B cell; Gr1: granulocyte; CD3: T cell; Mac1: monocyte; Ter119: erythrocyte). Most types of blood cells from mice transplanted with iRFP BM cells expressed iRFP. C: The expression of various blood cell markers in hematopoietic cells from WT and iRFP mice was similar. The data indicate as the means ± SEM. WT, n=3; iRFP mice, n=3.