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[17]. The use of a single episome for the high-level production of multiple genes has also proven somewhat difficult. When using the strong cytomegalovirus (CMV) immediate early promoter twice on one construct, we have encountered the phenomenon of promoter occlusion [18] in which transcription from one of the two promoters has been significantly dampened (R.A.H., unpublished observations). Furthermore, cloning multiple expression cassettes into one episome produces exceptionally large constructs that are cumbersome to generate and re-

Figure 2 Genomic Southern blot analysis. The lane contains DNA isolated from 293EBNA cells 8 weeks after transfection with pE3hyg containing the coding sequences of the rat Gia2 gene. The probe used, Gia2, illuminates 2 bands, one at 8.5 kb representing the genomic Gia2 copies endogenous to 293EBNA cells, and the other at 5.8 kb representing the episomal copies. The parental cell is hypotriploid with a modal chromosome number of 64 [15,16]. Therefore the genomic band is most likely derived from a triploid chromosome and represents three copies. Scanning densitometer measurements of several exposures indicate that the episomal band is represented at seven or eight copies per cell.

Figure 2 Genomic Southern blot analysis. The lane contains DNA isolated from 293EBNA cells 8 weeks after transfection with pE3hyg containing the coding sequences of the rat Gia2 gene. The probe used, Gia2, illuminates 2 bands, one at 8.5 kb representing the genomic Gia2 copies endogenous to 293EBNA cells, and the other at 5.8 kb representing the episomal copies. The parental cell is hypotriploid with a modal chromosome number of 64 [15,16]. Therefore the genomic band is most likely derived from a triploid chromosome and represents three copies. Scanning densitometer measurements of several exposures indicate that the episomal band is represented at seven or eight copies per cell.

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