Noninvasive in vivo monitoring of neuronal differentiation using reporter driven by a neuronal promoter.
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.
Hwang
Do Won
DW
Programs in Neuroscience, Seoul National University, Seoul, South Korea.
Kang
Joo Hyun
JH
Jeong
Jae Min
JM
Chung
June-Key
JK
Lee
Myung Chul
MC
Kim
Soonhag
S
Lee
Dong Soo
DS
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
D007457:Iodine Radioisotopes; D008156:Luciferases; D010751:Phosphopyruvate Hydratase; D027981:Symporters; D000099209:Sodium-Iodide Symporters