Long-term production of choline acetyltransferase in the CNS after transplantation of fibroblasts modified with a regulatable vector.
A rat fibroblast cell line was modified to contain the Drosophila choline acetyltransferase (ChAT) cDNA under the control of a tetracycline-regulated system. Several clonal lines were assessed in vitro and in vivo to establish the optimal clone for gene therapy experiments. The influence of in vitro cell density on ChAT expression was compared to biological activity detected after grafting to the rat brain. While each clone had different ChAT activity patterns, all clones had low activity immediately post-grafting which increased over time, reaching a plateau between 1 and 2 months which was maintained for at least 1 year. The clones expressed a high basal ChAT activity level in vitro that was repressed in a dose- and time-dependent manner with doxycycline (DOX) treatment. In the absence of DOX, high levels of ChAT activity were maintained for at least 2 months in vitro. DOX induced a rapid and strong (200-fold) suppression of ChAT activity within 48 h. A dose-response curve indicated that the fibroblasts were very sensitive to low concentrations of DOX (ED50 12 pg/ml). Removal of DOX led to a derepression of ChAT activity within 2 days. These cells will be useful for ex vivo gene therapy of the cholinergic system.
Pizzo
Donald P
DP
Department of Neurosciences, University of California San Diego, La Jolla, CA 92023, USA.
Paban
Veronique
V
Coufal
Nicole G
NG
Gage
Fred H
FH
Thal
Leon J
LJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D017690:Cell Transplantation; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D018678:Cholinergic Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology* / Q000637:transplantation*; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D000922:Immunotoxins / Q000494:pharmacology; D008297:Male; D009699:N-Glycosyl Hydrolases; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D051381:Rats; D011916:Rats, Inbred F344; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D013997:Time Factors
C085696:192 IgG-saporin; D000911:Antibodies, Monoclonal; D018678:Cholinergic Agents; D000922:Immunotoxins; D054789:Ribosome Inactivating Proteins, Type 1; D002795:Choline O-Acetyltransferase; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D004318:Doxycycline