title: pcDNA3.1tdTomato is superior to pDsRed2-N1 for optical fluorescence imaging in the F344/AY-27 rat model of bladder cancer.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  RESULTS
  AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY-27/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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, UK.
authors.forename: Vincent
authors.initials: V
authors.lastname: Koo
authors.forename: Alvin
authors.initials: A
authors.lastname: Lee
authors.forename: Osama Sharaf
authors.initials: OS
authors.lastname: Eldin
authors.forename: Chris
authors.initials: C
authors.lastname: Watson
authors.forename: Peter
authors.initials: P
authors.lastname: Hamilton
authors.forename: Kate
authors.initials: K
authors.lastname: Williamson
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D018744:DNA, Plant / Q000235:genetics*; D004195:Disease Models, Animal; D005453:Fluorescence; D018551:Solanum lycopersicum / Q000235:genetics*; D051381:Rats; D011916:Rats, Inbred F344; D055647:Rats, Transgenic; D001749:Urinary Bladder Neoplasms / Q000378:metabolism* / Q000473:pathology
chemical_list: D018744:DNA, Plant