`
`PCT/NL02/00390
`
`57
`
`ectopic expression in mammalian cell vectors. The Tet-Off promoter is
`
`inducible: the promoter is repressed in the presence of tetracycline or related
`
`antibiotics (doxycycline is commonly used) in cell-lines which express the tTA
`
`plasmid (Clontech K1620—A), and removal of the antibiotic results in
`
`5
`
`transcriptional induction (Deuschle et al., 1995, Gossen & Bujard, 1992, Izumi
`
`& Gilbert, 1999, Umana et al., 1999).
`
`Materials and Methods:
`
`10
`
`The construction of the pSDH-Tet and pSDH—CMV vectors is described in
`
`Example 11. pSDH—SV4O was constructed by PCR amplification of the SV4O
`
`promoter (primers D41 and D42) from plasmid pSelect—SV40-Zeo (Example 1),
`
`followed by digestion of the PCR product with SacII and SalI. The pSDH-CMV
`
`vector was digested with Sacll and SalI to remove the CMV promoter, and the
`
`15
`
`vector and SV4O fragment were ligated together to create pSDH-SV40. STARG
`
`was cloned into M081 and MOSH as described in Example 11. The plasmids
`
`pSDH~Tet, pSDH-Tet—STARB, pSDH—Tet—STAR7, pSDH-SV4O and pSDH-
`
`SV40-STAR6 were co-transfected with pBabe-Puro into U-2 OS using
`
`SuperFect as described by the manufacturer. Cell cultivation, puromycin
`
`20
`
`selection, and luciferase assays were carried out as described in Example 11.
`
`Results
`
`FIGS 9, 11, and 12 compare the expression of the luciferase reporter gene from
`
`25
`
`3 different promoters: two strong and constitutive viral promoters (CMV and
`
`SV40), and the inducible Tet~Off promoter. All three promoters were tested in
`
`the context of the STAR6 element in U—2 OS cells. The results demonstrate
`
`that the yield and predictability from all 8 promoters are increased by STARG.
`
`As described in Examples 11 and 14, STAR6 is beneficial in the context of the
`
`30
`
`CMV promoter (FIG 9). Similar improvements are seen in the context of the
`
`
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