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Int J Mol Sci. 2017 Dec 22;19(1). pii: E19. doi: 10.3390/ijms19010019.

Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo.

Author information

1
Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, UK. J.Zhou18@liverpool.ac.uk.
2
Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, UK. J.Sharkey@liverpool.ac.uk.
3
Centre for Preclinical Imaging, University of Liverpool, Liverpool L69 3GE, UK. J.Sharkey@liverpool.ac.uk.
4
Alder Hey Children's NHS Foundation Trust, Liverpool L12 2AP, UK. Rajeev.Shukla@alderhey.nhs.uk.
5
Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, UK. A.Plagge@liverpool.ac.uk.
6
Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, UK. P.A.Murray@liverpool.ac.uk.
7
Centre for Preclinical Imaging, University of Liverpool, Liverpool L69 3GE, UK. P.A.Murray@liverpool.ac.uk.

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.

KEYWORDS:

E2-Crimson; fluorescent reporter; in vivo imaging; knock-in; mouse embryonic stem cells

PMID:
29271879
PMCID:
PMC5795970
DOI:
10.3390/ijms19010019
[Indexed for MEDLINE]
Free PMC Article

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