Abstract
Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C), for tracking mouse embryonic cells (mESCs) in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over nine days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus.
Highlights
During the past two decades, considerable advances have been made in the field of fluorescent imaging technology, especially with regard to fluorescent reporter genes, which are useful because they facilitate long-term cell tracking
Targeted integration at the Rosa26 locus generally introduces only a single copy of the transgene into the Rosa26 allele [27], leading to lower expression levels than what can be achieved with lentiviral transduction, there is a lower tendency for transgenes within the Rosa26 locus to become silenced following differentiation of the Mouse embryonic stem cells (mESCs) [15]
The main conclusion from this experiment is that the E2C is not an effective in vivo reporter in this context, and imaging the E2C reporters in the internal organs would not be feasible due to signal attenuation with increasing depth
Summary
During the past two decades, considerable advances have been made in the field of fluorescent imaging technology, especially with regard to fluorescent reporter genes, which are useful because they facilitate long-term cell tracking. Cell nuclei and mitochondria cause light scattering within the optical window [2], whereas the intensity can be reduced by using longer wavelengths [3]. This requires fluorescent probes that have emission spectra within the far-red region (710–850 nm wavelength, which is part of the near-infrared spectrum). The excitation and emission maxima of E2C are 611 and 646 nm, respectively This means that it can be excited efficiently by standard far-red lasers routinely used in optical instruments, making it suitable for tracking cells in vivo [4]
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