Figure 1.

Doxycycline-inducible adeno-associated virus (AAV) vectors. (a) AAV vectors plasmids used in this study. pAC1-M2-GDNF (previously described³¹) is an autoregulated tetracycline-inducible vector haboring a central bidirectional tetracycline-responsive promoter directing transcription of both the human GDNF cDNA and the M2 mutant of the reverse tetracycline transactivator (rtTA).²³ In pAC1-M2-LUC, the hGDNF cDNA has been replaced by the LUC reporter gene (from pBI-GL, GenBank Accession No. U89935). In pAC1-V16 vector, the rtTA M2 mutant has been replaced by the V16 mutant.²⁰ ITR, AAV inverted terminal repeat; pTet_bidi, bidirectional tetracycline-responsive promoter promoter (from pBI-GL, GenBank Accession No. U89935); rtTA-M2, reverse tetracycline transactivator mutant; rtTA-V16, reverse tetracycline transactivator mutant; bidirectional arrow, bidirectional SV40 polyadenylation site. (b,c) In vitro comparison of Dox sensitivity. HEK-293T cells were coinfected with pAC1 constructs encapsidated into serotype 1 AAV capsids and adenovirus type 5 helper. Cells were treated with Dox at increasing doses for 48 hours. For glial cell line-derived neurotrophic factor (GDNF) vectors, the medium was harvested 48 hours postinfection and analyzed using an enzyme-linked immunosorbent assay. For LUC vectors, cells were lysed and protein extracts analyzed using a luminometer. RLU, relative light units. *P < 0.05; **P < 0.01; ***P < 0.001 versus untreated cells.