Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators

Stefania Lamartina, Carlo Toniatti, Giuseppe Roscilli, Cira Daniela Rinaudo, Elisabetta Sporeno, Luisa Silvi, Wolfgang Hillen, Hermann Bujard, Riccardo Cortese, Gennaro Ciliberto

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2S-S2 and rtTA2S-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2S-M2 and rtTA2S-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 μg of a single plasmid containing the rtTA2S-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2S-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

Original languageEnglish
Pages (from-to)199-210
Number of pages12
JournalHuman Gene Therapy
Volume13
Issue number2
DOIs
Publication statusPublished - 2002

Fingerprint

Trans-Activators
Doxycycline
Tetracycline
Gene Expression
Plasmids
Erythropoietin
Hematocrit
Quadriceps Muscle
Genetic Therapy
Complementary DNA
Ligands
Cell Line
DNA
Genes

ASJC Scopus subject areas

  • Genetics

Cite this

Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators. / Lamartina, Stefania; Toniatti, Carlo; Roscilli, Giuseppe; Rinaudo, Cira Daniela; Sporeno, Elisabetta; Silvi, Luisa; Hillen, Wolfgang; Bujard, Hermann; Cortese, Riccardo; Ciliberto, Gennaro.

In: Human Gene Therapy, Vol. 13, No. 2, 2002, p. 199-210.

Research output: Contribution to journalArticle

Lamartina, S, Toniatti, C, Roscilli, G, Rinaudo, CD, Sporeno, E, Silvi, L, Hillen, W, Bujard, H, Cortese, R & Ciliberto, G 2002, 'Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators', Human Gene Therapy, vol. 13, no. 2, pp. 199-210. https://doi.org/10.1089/10430340252769734
Lamartina, Stefania ; Toniatti, Carlo ; Roscilli, Giuseppe ; Rinaudo, Cira Daniela ; Sporeno, Elisabetta ; Silvi, Luisa ; Hillen, Wolfgang ; Bujard, Hermann ; Cortese, Riccardo ; Ciliberto, Gennaro. / Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators. In: Human Gene Therapy. 2002 ; Vol. 13, No. 2. pp. 199-210.
@article{86441aa5af354db7a114f17a4fb7eaf9,
title = "Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators",
abstract = "The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2S-S2 and rtTA2S-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2S-M2 and rtTA2S-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 μg of a single plasmid containing the rtTA2S-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2S-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.",
author = "Stefania Lamartina and Carlo Toniatti and Giuseppe Roscilli and Rinaudo, {Cira Daniela} and Elisabetta Sporeno and Luisa Silvi and Wolfgang Hillen and Hermann Bujard and Riccardo Cortese and Gennaro Ciliberto",
year = "2002",
doi = "10.1089/10430340252769734",
language = "English",
volume = "13",
pages = "199--210",
journal = "Human Gene Therapy",
issn = "1043-0342",
publisher = "Mary Ann Liebert Inc.",
number = "2",

}

TY - JOUR

T1 - Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators

AU - Lamartina, Stefania

AU - Toniatti, Carlo

AU - Roscilli, Giuseppe

AU - Rinaudo, Cira Daniela

AU - Sporeno, Elisabetta

AU - Silvi, Luisa

AU - Hillen, Wolfgang

AU - Bujard, Hermann

AU - Cortese, Riccardo

AU - Ciliberto, Gennaro

PY - 2002

Y1 - 2002

N2 - The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2S-S2 and rtTA2S-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2S-M2 and rtTA2S-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 μg of a single plasmid containing the rtTA2S-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2S-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

AB - The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2S-S2 and rtTA2S-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2S-M2 and rtTA2S-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 μg of a single plasmid containing the rtTA2S-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2S-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

UR - http://www.scopus.com/inward/record.url?scp=18244382853&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=18244382853&partnerID=8YFLogxK

U2 - 10.1089/10430340252769734

DO - 10.1089/10430340252769734

M3 - Article

C2 - 11812277

AN - SCOPUS:18244382853

VL - 13

SP - 199

EP - 210

JO - Human Gene Therapy

JF - Human Gene Therapy

SN - 1043-0342

IS - 2

ER -