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Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking.
J Nucl Med 2005; 46(2):305-11JN

Abstract

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.

CONCLUSION

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.

Authors+Show Affiliations

Program in Neuroscience, Seoul National University College of Natural Science, Seoul 110-744, Korea.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Studies
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15695791

Citation

Kim, Yun Hui, et al. "Reversing the Silencing of Reporter Sodium/iodide Symporter Transgene for Stem Cell Tracking." Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine, vol. 46, no. 2, 2005, pp. 305-11.
Kim YH, Lee DS, Kang JH, et al. Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking. J Nucl Med. 2005;46(2):305-11.
Kim, Y. H., Lee, D. S., Kang, J. H., Lee, Y. J., Chung, J. K., Roh, J. K., ... Lee, M. C. (2005). Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking. Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine, 46(2), pp. 305-11.
Kim YH, et al. Reversing the Silencing of Reporter Sodium/iodide Symporter Transgene for Stem Cell Tracking. J Nucl Med. 2005;46(2):305-11. PubMed PMID: 15695791.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking. AU - Kim,Yun Hui, AU - Lee,Dong Soo, AU - Kang,Joo Hyun, AU - Lee,Young Jin, AU - Chung,June-Key, AU - Roh,Jae-Kyu, AU - Kim,Seung Up, AU - Lee,Myung Chul, PY - 2005/2/8/pubmed PY - 2005/4/9/medline PY - 2005/2/8/entrez SP - 305 EP - 11 JF - Journal of nuclear medicine : official publication, Society of Nuclear Medicine JO - J. Nucl. Med. VL - 46 IS - 2 N2 - UNLABELLED: 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. CONCLUSION: 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. SN - 0161-5505 UR - https://www.unboundmedicine.com/medline/citation/15695791/Reversing_the_silencing_of_reporter_sodium/iodide_symporter_transgene_for_stem_cell_tracking_ L2 - http://jnm.snmjournals.org/cgi/pmidlookup?view=long&pmid=15695791 DB - PRIME DP - Unbound Medicine ER -