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



Zhou, Jing, Sharkey, Jack, Shukla, Rajeev, Plagge, Antonius ORCID: 0000-0001-6592-1343 and Murray, Patricia ORCID: 0000-0003-1316-148X
(2018) Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 19 (1). E19-.

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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 <i>E2C</i> into the <i>Rosa26</i> locus of an <i>E14-Bra-GFP</i> 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 <i>Rosa26</i> locus.

Item Type: Article
Uncontrolled Keywords: fluorescent reporter, E2-Crimson, mouse embryonic stem cells, knock-in, in vivo imaging
Depositing User: Symplectic Admin
Date Deposited: 02 Jan 2018 10:31
Last Modified: 02 Jan 2024 09:56
DOI: 10.3390/ijms19010019
Open Access URL: http://www.mdpi.com/1422-0067/19/1/19
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URI: https://livrepository.liverpool.ac.uk/id/eprint/3014990