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 language | English |
|---|---|
| Pages (from-to) | 199-210 |
| Number of pages | 12 |
| Journal | Human Gene Therapy |
| Volume | 13 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2002 |
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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 journal › Article
}
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 -