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Mammalian and Bacterial Expression in One Vector


Novel pdual expression vector for high-level expression in both mammalian and bacterial cells

Kerstien A. Padgett Joseph A. Sorge
Stratagene

Stratagene has developed the pdual expression vector,* which permits high-level expression of heterologous genes in both eukaryotic and prokaryotic systems. The vector contains the promoter and enhancer region of the human cytomegalovirus (CMV) immediate early gene for constitutive expression of cloned DNA inserts in mammalian cells. Expression of heterologous genes in bacteria is regulated by a hybrid T7/ lacO promoter. The pDual vector was engineered to carry the lac repressor gene (lacI). Expression is therefore inducible using IPTG in bacteria that contain the T7 RNA polymerase. ## In both bacterial and mammalian cells, the dominant selectable marker is the neomycin phosphotransferase gene under the dual control of the b-lactamase and SV40 promoters. A distinguishing feature of the pDual vector is the tandemly arranged bacterial Shine-Dalgarno1 and mammalian Kozak 2 consensus sequence. Each ribosome binding site is positioned at its optimal distance from the initiation codon of the target gene, which ensures efficient translation of mRNA generated in either system.

Mammalian expression vectors contain similar elements that may also be present in bacterial expression vectors. Yet mammalian and bacterial plasmid vectors vary considerably in their choice of promoters, the presence of splice signals and polyadenylation sites, as well as their respective translation initiation sequences. In fact, the differences between bacterial and mammalian expression vectors are so significant that, until now, these vectors could not be used interchangeably.

For expression in mammalian cells, eukaryotic genes are typically cloned first into
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