`
`PCT/NL02/00390
`
`59
`
`must be considered. This example compares the native orientation with the
`
`opposite orientation (FIG 13).
`
`Materials and Methods:
`
`The STAR66 element 'was cloned into pSDI—I-Tet as described in Example 11.
`
`U-2 OS cells were co-transfected with plasmids pSDI-I—Tet-STAR66-native and
`
`pSDH—Tet-STARBB-opposite, and cultivated as described in Example 11.
`
`Individual clones were isolated and cultivated; the level of luciferase
`
`expression was determined as described (supra).
`
`Results
`
`The results of the comparison of STARGG activity in the native orientation and
`
`the opposite orientation are shown in FIG 14. When STAR66 is in the opposite
`
`orientation, the yield of only one clone is reasonably high (60 luciferase units).
`
`In contrast, the yield of the highest—expressing clone when STAR66 is in the
`
`native orientation is considerably higher (100 luciferase units), and the
`
`predictability is much higher as well: 7 clones of the native-orientation
`
`population (30%) express luciferase above the level of the highest-expressing
`
`clone from the opposite-orientation population, and 15 of the clones in the
`
`native—orientation population (60%) express luciferase above 10 relative
`
`luciferase units. Therefore it is demonstrated that STAR66 function is
`
`directional.
`
`Example 17
`
`Transgene expression in the context of STAR elements is copy number-
`
`dependent
`
`Transgene expression units for heterologous protein expression are generally
`
`integrated into the genome of the host cell to ensure stable retention during
`
`cell division. Integration can result in one or multiple copies of the expression
`
`10
`
`15
`
`20
`
`25
`
`30
`
`
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