Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites.
The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture.
Tornøe
Jens
J
NsGene A/S, Pederstrupvej 93, DK-2750, Ballerup, Denmark. jt@nsgene.dk
Kusk
Philip
P
Johansen
Teit E
TE
Jensen
Peter R
PR
D000199:Actins / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D015870:Gene Expression; D015723:Gene Library; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D025822:Ubiquitin C / Q000235:genetics
D000199:Actins; D008164:Luminescent Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D025822:Ubiquitin C; D049452:Green Fluorescent Proteins