High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.
Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan. kanegae@ims.u-tokyo.ac.jp
Yumi
Y
Kanegae
Miho
M
Terashima
Saki
S
Kondo
Hiromitsu
H
Fukuda
Aya
A
Maekawa
Zheng
Z
Pei
Izumu
I
Saito
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000235:genetics
D034741:RNA, Small Interfering; D000509:alpha-Fetoproteins; C045073:Cre recombinase; D019426:Integrases