CS2+ is a multipurpose expression vector. Although primarily designed for expressing proteins in Xenopus embryos from either injected RNA or DNA, CS2+ is also useful for high-level transient expression in a wide variety of mammalian and avian cells. It is also functional in zebrafish embryos (as DNA or RNA), and it can be used for in vitro transcription/translation (using, for example, the Promega TnT system).
CS2+ features: CS2+ contains a strong enhancer/promoter (simian CMV IE94) followed by a polylinker and the SV40 late polyadenlyation site. An SP6 promoter is present in the 5' untranslated region of the mRNA from the sCMV promoter, allowing in vitro RNA synthesis of sequences cloned into the polylinker. A T7 promoter in reverse orientation between the polylinker and the SV40 polyA site for probe synthesis, as well as a second polylinker after the SV40 polyA site to provide several possible sites to linearize the vector for SP6 RNA transcription. The vector backbone is from pBluescript II KS+ and includes the ampr gene and an f1 origin for single stranded DNA.
Improved protein yields in Xenopus embryos using CS2 synthetic RNAs: We have observed that capped SP6 mRNAs from CS2 which include the SV40 polyA site at the 3' end (DNA templates linearized at polylinker II, usually with Asp718 or Not1) are more stable than conventional capped RNAs when injected into Xenopus embryos. In addition, these mRNAs produce substantially more protein than conventional mRNAs (10X or more in many cases, R. Rupp, L. Snider, and D. Turner, unpublished). These effects appear to reflect polyadenylation of the injected CS2 mRNA. We do not know if CS2 SP6 mRNAs are polyadenylated in zebrafish, Xenopus oocytes, or other systems.
CS2 derivatives: A number of derivatives of CS2 exist (see list next page). These include versions that are designed to express proteins as fusions with 6 copies of the myc epitope tag (with or without a nuclear localization sequence [NLS]), a version for expressing proteins fused to an NLS, and versions that express either cytoplasmic or nuclear localized b-galactosidase (also potential fusion proteins). Derivatives are identical to CS2+ outside the polylinker I region, except for CS2- (f1 origin reversed) and CS2+IREScbgal.
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