hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo.
To advance our understanding of the central nervous system, there is a need for refined approaches to control gene expression in neuronal culture as well as in the brain in vivo. In this study, we have applied a doxycycline-responsive Tet system to obtain a tightly controlled gene expression in neurons. In the absence of doxycycline, the Tet promoter-driven transgene expression was blocked by Tet transrepressor (tTR). Expression was doxycycline activated with the aid of a reverse Tet transactivator (rtTA). Application of both tTR and rtTA resulted in a much greater inducibility, as compared to rtTA alone, mainly due to a decreased basal level of expression. Such effects were observed when tTR and rtTA were driven in cultured neurons by the alpha CaMKII promoter. However, introduction of the human CMV major immediate-early promoter resulted only in a mediocre neuronal gene expression, unless the cells were treated, either in culture or in vivo, with depolarizing concentrations of KCl. Thus, in the present report, we have examined hCMV and Tet promoter inducibility in neurons to produce an important improvement in the functioning of the Tet system.
Konopka
Witold
W
Department of Molecular and Cellular Neurobiology, Nencki Institute, Pasteura 3, 02-093 Warsaw, Poland.
Duniec
Kamila
K
Mioduszewska
Barbara
B
Proszynski
Tomasz
T
Jaworski
Jacek
J
Kaczmarek
Leszek
L
D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008297:Male; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
D012097:Repressor Proteins; D015534:Trans-Activators; C034811:tetracycline resistance-encoding transposon repressor protein