In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

Chia Hung Hsieh, Ren Shyan Liu, Hsin Ell Wang, Jeng Jong Hwang, Win Ping Deng, Jyh Cheng Chen, Fu Du Chen

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [131I] FIAU and [3H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by inducible promoters or a less stable protein with a more rapid turnover due to the limitations of the half-life of the HSV1-TK enzyme and the cellular retention time of their phosphorylated molecular probes.

Original languageEnglish
Pages (from-to)653-660
Number of pages8
JournalNuclear Medicine and Biology
Volume33
Issue number5
DOIs
Publication statusPublished - Jul 2006

Fingerprint

Thymidine Kinase
Human Herpesvirus 1
Genes
Gene Expression
Tetracycline
Half-Life
Molecular Probes
Doxycycline
Luciferases
Reporter Genes
Cell Line
Firefly Luciferases
In Vitro Techniques
Enzyme Stability
Proteins
Enzyme Assays
Protein Transport
Enzymes
Cycloheximide
Radioisotopes

Keywords

  • [I] FIAU
  • [H] GCV
  • Gene expression imaging
  • Herpes simplex virus thymidine kinase
  • Luciferase

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Medicine
  • Radiology Nuclear Medicine and imaging

Cite this

In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation. / Hsieh, Chia Hung; Liu, Ren Shyan; Wang, Hsin Ell; Hwang, Jeng Jong; Deng, Win Ping; Chen, Jyh Cheng; Chen, Fu Du.

In: Nuclear Medicine and Biology, Vol. 33, No. 5, 07.2006, p. 653-660.

Research output: Contribution to journalArticle

Hsieh, Chia Hung ; Liu, Ren Shyan ; Wang, Hsin Ell ; Hwang, Jeng Jong ; Deng, Win Ping ; Chen, Jyh Cheng ; Chen, Fu Du. / In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation. In: Nuclear Medicine and Biology. 2006 ; Vol. 33, No. 5. pp. 653-660.
@article{ab4672e313fe4b579472263766972fbe,
title = "In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation",
abstract = "The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [131I] FIAU and [3H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by inducible promoters or a less stable protein with a more rapid turnover due to the limitations of the half-life of the HSV1-TK enzyme and the cellular retention time of their phosphorylated molecular probes.",
keywords = "[I] FIAU, [H] GCV, Gene expression imaging, Herpes simplex virus thymidine kinase, Luciferase",
author = "Hsieh, {Chia Hung} and Liu, {Ren Shyan} and Wang, {Hsin Ell} and Hwang, {Jeng Jong} and Deng, {Win Ping} and Chen, {Jyh Cheng} and Chen, {Fu Du}",
year = "2006",
month = "7",
doi = "10.1016/j.nucmedbio.2006.04.001",
language = "English",
volume = "33",
pages = "653--660",
journal = "Nuclear Medicine and Biology",
issn = "0969-8051",
publisher = "Elsevier Inc.",
number = "5",

}

TY - JOUR

T1 - In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

AU - Hsieh, Chia Hung

AU - Liu, Ren Shyan

AU - Wang, Hsin Ell

AU - Hwang, Jeng Jong

AU - Deng, Win Ping

AU - Chen, Jyh Cheng

AU - Chen, Fu Du

PY - 2006/7

Y1 - 2006/7

N2 - The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [131I] FIAU and [3H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by inducible promoters or a less stable protein with a more rapid turnover due to the limitations of the half-life of the HSV1-TK enzyme and the cellular retention time of their phosphorylated molecular probes.

AB - The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [131I] FIAU and [3H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by inducible promoters or a less stable protein with a more rapid turnover due to the limitations of the half-life of the HSV1-TK enzyme and the cellular retention time of their phosphorylated molecular probes.

KW - [I] FIAU

KW - [H] GCV

KW - Gene expression imaging

KW - Herpes simplex virus thymidine kinase

KW - Luciferase

UR - http://www.scopus.com/inward/record.url?scp=33745806870&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33745806870&partnerID=8YFLogxK

U2 - 10.1016/j.nucmedbio.2006.04.001

DO - 10.1016/j.nucmedbio.2006.04.001

M3 - Article

C2 - 16843840

AN - SCOPUS:33745806870

VL - 33

SP - 653

EP - 660

JO - Nuclear Medicine and Biology

JF - Nuclear Medicine and Biology

SN - 0969-8051

IS - 5

ER -