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Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells

of the CMV promoter; and the CMV promoter in this construct can be readily replaced with other promoters to confer cell-type specificity to receptor expressionan advantage that is particularly attractive for the construction of transgenic animals. The plasmid also contains a neomycin-resistance gene, which is expressed in both E. coli (kanamycin-resistance) and mammalian cells (G418-resistance).

Fig.2

The ecdysone-inducible expression vector pEGSH contains the ponA-inducible expression cassette comprised of 5 E/GRE binding sites upstream of a minimal promoter consisting of three SP1 sites, followed by the D. melanogaster hsp27 minimal promoter (Figure 2B ). The vector contains the hygromycin-resistance gene to allow stable selection in cells transformed with the pERV3 plasmid. The MCS of pEGSH was engineered to contain an array of restriction sites positioned for directional cloning of inserts derived from Lambda ZAP-derived cDNA vectors*, lgt10, lgt11, HybriZAP vectors, and other two-hybrid libraries, in addition to most other popular cDNA cloning and expression vectors.

In addition to this versatile array of restriction sites, the pEGSH MCS contains the following features: the ability to monitor expression of the gene of interest, by either a-FLAG immunodetectionll ll or by RNA detection using T3 antisense RNA probes; and the ability to seamlessly fuse the insert to the FLAG epitope and/or the HSP leader by using the seamless cloning kit** for which 80% cloning efficiencies are routinely achieved.

Transient Expression Assays

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