Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking.
To track neural stem cells transfected with reporter gene, perpetual stem cell transgene expression is required. Referring to the knowledge about epigenetic modulation, we succeeded in reversing the silencing of sodium/sodide symporter (hNIS) transgenes transfected into human neural stem (HB1.F3) cells.


METHODS
hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of cytomegalovirus promoter, and this construct was transfected into HB1.F3 cells to yield the F3-NIS cell lines. hNIS transgene expression was examined by (125)I uptake and reverse transcriptase polymerase chain reaction (RT-PCR). The iodide uptake of F3-NIS III cells was initially higher by up to 12.9-fold than that of nontransfected HB1.F3 cells. However, repeated passage gradually silenced hNIS expression. The recovery of hNIS transgene expression by demethylating agent (5-azacytidine) or histone deacetylase inhibitor (trichostatin A; TSA) treatment was investigated.


RESULTS
As hNIS transgene was gradually silenced in F3-NIS III cells, after the eighth passage its iodide uptake was 1.9-fold higher than that of nontransfected HB1.F3 cells. 5-azacytidine treatment (up to 40 micromol/L) for 24 h in F3-NIS III cells increased iodide uptake and hNIS messenger RNA (mRNA) 1.8- and 1.9-fold versus nontreated F3-NIS cells, respectively. Moreover, after TSA treatment (up to 62.5 nmol/L) for 24 h, iodide uptake and hNIS mRNA in F3-NIS III cells increased 36- and 1.9-fold versus nontreated F3-NIS III cells, respectively. The synergistic effect of demethylation and histone deacetylation inhibition was significant at high-dose 5-azacytidine and low-dose of TSA treatment. After treating F3-NIS III cells in vitro for 24 h with 62.5 nmol/L TSA, the cells were implanted into BALB/c nude mice. The TSA-treated F3-NIS III cells were clearly visible on gamma-camera imaging using (99m)Tc-pertechnetate as compared with F3-NIS III cells not treated with TSA.


CONCLUSIONS
These results suggest that 2 well-known mechanisms of epigenetic modulation synergistically are involved in silencing reporter hNIS transgene in a neural stem cell line. Transgene silencing was reversed using demethylation and histone deacetylation inhibition. We conclude that silenced reporter transgenes once successfully expressed in stem cells might be awakened by pharmacologic treatment before infusion to track stem cells in vivo.
Kim
Yun Hui
YH
Program in Neuroscience, Seoul National University College of Natural Science, Seoul 110-744, Korea.
Lee
Dong Soo
DS
Kang
Joo Hyun
JH
Lee
Young Jin
YJ
Chung
June-Key
JK
Roh
Jae-Kyu
JK
Kim
Seung Up
SU
Lee
Myung Chul
MC
D000818:Animals; D002452:Cell Count / Q000379:methods*; D002465:Cell Movement / Q000502:physiology*; D002469:Cell Separation / Q000379:methods*; D020868:Gene Silencing; D006801:Humans; D007457:Iodine Radioisotopes / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D015202:Protein Engineering / Q000379:methods; D011877:Radionuclide Imaging; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000000981:diagnostic imaging* / Q000502:physiology*; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics; D000099209:Sodium-Iodide Symporters
D007457:Iodine Radioisotopes; D027981:Symporters; D000099209:Sodium-Iodide Symporters