title: Noninvasive in vivo monitoring of neuronal differentiation using reporter driven by a neuronal promoter.
abstract: OBJECTIVE
  We imaged neuronal differentiation in vivo using dual reporters (sodium iodide symporter [NIS] and luciferase) coupled to a neuron-specific enolase (NSE) promoter.
  
  
  METHODS
  PC12 (NSE positive) and F11 cells were transfected with a bicistronic (NIS and luciferase; pNSE-NF) or a luciferase (pNSE-Fluc) reporter coupled to the NSE promoter. Weak NSE promoter activity was overcome by a two-step transcriptional amplification (TSTA) system (pNSE-TSTA-Fluc). In vivo, NIS and luciferase expression were examined using a (99m)Tc-pertechnetate gamma camera and bioluminescence imaging, respectively.
  
  
  RESULTS
  pNSE-NF-transfected PC12 cells showed 3-fold higher radioiodine uptakes and >100-fold higher luciferase activity than parental cells. NIS or luciferase activity was not detected in pNSE-NF-transfected HeLa cells. When F11 cells were differentiated into neurons by db-cAMP, NIS and luciferase activities increased 4-fold compared to those without treatment, which was confirmed by Western blot and RT-PCR of NSE. In vivo in pNSE-NF-transfected F11 cells, db-cAMP treatment increased the luciferase activity but not the scintigraphic activity. In vitro, pNSE-TSTA-Fluc produced 130-fold higher luciferase activity than pNSE-Fluc and neuronal differentiation showed 4-fold higher activity from both pNSE-TSTA-Fluc and pNSE-Fluc than before differentiation. In vivo, in pNSE-TSTA-Fluc-transfected F11 cells, luciferase activity increased after neuronal differentiation. In vivo luciferase activity persisted up to 2 days after db-cAMP-induced neuronal differentiation.
  
  
  CONCLUSIONS
  NSE promoter-driven dual reporter transgenes revealed the possibility of in vivo imaging of neuronal differentiation, which was further enabled by high amplification using a TSTA system. We propose that this strategy be used to follow the transplanted stem cells during differentiation in live animals.
authors: Programs in Neuroscience, Seoul National University, Seoul, South Korea.
authors: Do Won
authors: DW
authors: Hwang
authors: Joo Hyun
authors: JH
authors: Kang
authors: Jae Min
authors: JM
authors: Jeong
authors: June-Key
authors: JK
authors: Chung
authors: Myung Chul
authors: MC
authors: Lee
authors: Soonhag
authors: S
authors: Kim
authors: Dong Soo
authors: DS
authors: Lee
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007457:Iodine Radioisotopes; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D049449:Luminescence; D009474:Neurons / Q000166:cytology* / Q000000981:diagnostic imaging / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011877:Radionuclide Imaging; D051381:Rats; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099209:Sodium-Iodide Symporters
chemical_list: D007457:Iodine Radioisotopes; D008156:Luciferases; D010751:Phosphopyruvate Hydratase; D027981:Symporters; D000099209:Sodium-Iodide Symporters