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B. Use of Single vs. Multiple Episomes

Among targets for drug discovery, the need to express two or more recombinant proteins is frequently encountered. For example, numerous receptors, transporters, and ion channels are composed of multiple subunits that must be present in stoichiometric quantities for functional activity. In addition, signal transduction cascades contain many potential targets of interest for drug intervention. Traditionally, integrating vectors have been used in order to express multiple gene products. The cointegration and concomitant coexpression with desired stoichi-ometry of two or more genes is a rare, labor-intensive event to obtain and exploit

Figure 1 Northern blot analysis. Concentration of steady-state RNA of CAM, a recombinant cell adhesion molecule, is compared to the housekeeping gene, GAPDH (glyceral-dehyde-3-phosphate-dehydrogenase). RNA from the hygromycin resistance marker (Hyg) is also visible. CAM, transcribed from a CMV promoter, and hyg, transcribed from a weak herpes simplex virus thymidine kinase (tk) promoter, are both contained on the episomal vector. Numbers to the left of the figure correspond to transcript length (in kilobases). Lanes A and B represent RNA isolated from untransfected and transfected 293EBNA cells (Invitrogen), respectively. Based on densitometer scans of several different exposures, the RNAs in this figure are represented at approximately the following ratios: 2:1: <0.1 for CAM:GAPDH:Hyg, respectively.

Figure 1 Northern blot analysis. Concentration of steady-state RNA of CAM, a recombinant cell adhesion molecule, is compared to the housekeeping gene, GAPDH (glyceral-dehyde-3-phosphate-dehydrogenase). RNA from the hygromycin resistance marker (Hyg) is also visible. CAM, transcribed from a CMV promoter, and hyg, transcribed from a weak herpes simplex virus thymidine kinase (tk) promoter, are both contained on the episomal vector. Numbers to the left of the figure correspond to transcript length (in kilobases). Lanes A and B represent RNA isolated from untransfected and transfected 293EBNA cells (Invitrogen), respectively. Based on densitometer scans of several different exposures, the RNAs in this figure are represented at approximately the following ratios: 2:1: <0.1 for CAM:GAPDH:Hyg, respectively.

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