pcDNA3.1tdTomato Is Superior to pDsRed2-N1 for Optical Fluorescence Imaging in the F344/AY-27 Rat Model of Bladder Cancer
Animal models are important for pre-clinical assessment of novel therapies in metastatic bladder cancer. The F344/AY-27 model involves orthotopic colonisation with AY-27 tumour cells which are syngeneic to F344 rats. One disadvantage of the model is the unknown status of colonisation between instillation and sacrifice. Non-invasive optical imaging using red fluorescence reporters could potentially detect tumours in situ and would also reduce the number of animals required for each experiment.
Materials and Methods
AY-27 cells were stably transfected with either pDsRed2-N1 or pcDNA3.1tdTomato. The intensity and stability of fluorescence in the resultant AY-27/DsRed2-N1 and AY-27/tdTomato stable cell lines were compared using Xenogen IVIS®200 and Olympus IX51 systems.
AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY27/DsRed2-N1 and was sustained in AY-27/tdTomato cells following freezing and six subsequent sub-cultures. After sub-cutaneous injection, fluorescence intensity from AY-27/tdTomato cells was threefold stronger than that detected from AY-27/DsRed2-N1 cells. IVIS®200 detected fluorescence from AY-27/tdTomato and AY-27/DsRed2-N1 cells colonising resected and exteriorised bladders, respectively. However, the deep-seated position of the bladder precluded in vivo imaging. Characteristics of AY-27/tdTomato cells in vitro and in tumours colonising F344 rats resembled those of parental AY-27 cells. Tumour transformation was observed in the bladders colonised with AY-27/DsRed2-N1 cells.
In vivo whole-body imaging of internal red fluorescent animal tumours should use pcDNA3.1tdTomato rather than pDsRed2-N1. Optical imaging of deep-seated organs in larger animals remains a challenge which may require proteins with brighter red or far-red fluorescence and/or alternative approaches.
Key wordsFluorescence DsRed2-N1 tdTomato IVIS®200 imaging AY-27 bladder tumour Rat model
The authors thank Professor Roger Tsien (University California of San Diego, USA) for the kind gift of the tdTomato gene in pRSET-B vector. This project has been funded by Research and Development Offices, HPSS Northern Ireland (AL); Mason’s Medical Foundation (VK); Egyptian Cultural Bureau (OS); Department of Employment and Learning (CW). We would like to thank Mr. Ken Arthur for his help with DNA flow cytometry.
All animal studies described in this manuscript were conducted according to Animal Licencing guidelines and approval.
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