Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo
Abstract
:1. Introduction
2. Results
2.1. Rosa26 Knock-in of E2-Crimson Transgene and Analysis of E2C Expression in Screened Positive Clones
2.2. Stemness and Differentiation Potential of Bra-GFP/Rosa26-E2C mESC Reporter Line
2.3. Quantitative Analysis of E2C Fluorescence Signal In Vivo
2.4. Histopathology and Immunofluorescence Analyses of Tumours
3. Discussion
4. Materials and Methods
4.1. Sub-Culture of STO and mESCs
4.2. Preparation of Mitomycin-C-Inactivated STO Feeder Cells
4.3. Preparation and Linearisation of Knock-in Construct
4.4. Generation of the Knock-in mESC Reporter Line
4.5. Genomic DNA Extraction of mESCs
4.6. 3′-Homology Arm PCR Analysis
4.7. Analysis of E2C Expression in the PCR Screened Positive Clones with Fluorescence Microscopy
4.8. Flow Cytometry Analysis
4.9. Embryoid Body Formation
4.10. Administration and Imaging of Bra-GFP/Rosa26-E2C
4.11. Tumour Volume Measurement
4.12. Tumour Fixation
4.13. Tumour Histopathological Analysis
4.14. Immunofluorescence Staining of Stemness Markers
4.15. Immunofluorescence Staining of Frozen Tumour Sections
4.16. Statistical Analysis
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
DAPI | 4′,6-diamidino-2-phenylindole |
DT-A | Diphtheria toxin A |
E2C | E2-crimson |
EB | Embryoid body |
FBS | Foetal bovine serum |
HA | Homology arm |
H&E | Haematoxylin and eosin |
mESC | Mouse embryonic stem cell |
PBS | Phosphate-buffered saline |
PECAM-1 | Platelet endothelial cell adhesion molecule |
PFA | Paraformaldehyde |
ROSA | Reverse orientation splice acceptor |
SCID | Severe combined immunodeficient |
Appendix A
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Zhou, J.; Sharkey, J.; Shukla, R.; Plagge, A.; Murray, P. Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo. Int. J. Mol. Sci. 2018, 19, 19. https://doi.org/10.3390/ijms19010019
Zhou J, Sharkey J, Shukla R, Plagge A, Murray P. Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo. International Journal of Molecular Sciences. 2018; 19(1):19. https://doi.org/10.3390/ijms19010019
Chicago/Turabian StyleZhou, Jing, Jack Sharkey, Rajeev Shukla, Antonius Plagge, and Patricia Murray. 2018. "Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo" International Journal of Molecular Sciences 19, no. 1: 19. https://doi.org/10.3390/ijms19010019