title: Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter.
abstract: Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.
authors.affiliation: Department of Molecular Biology, Research Institute of Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Takefumi
authors.initials: T
authors.lastname: Sone
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yahata
authors.forename: Hiroe
authors.initials: H
authors.lastname: Kishine
authors.forename: Junko
authors.initials: J
authors.lastname: Hotta
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
authors.forename: Fumio
authors.initials: F
authors.lastname: Imamoto
mesh_terms: D051344:Actin Capping Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D016366:Open Reading Frames / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D051344:Actin Capping Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D014764:Viral Proteins