( ction of PonA-Induced Gene Expression ...)Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells

Current commercially available systems lack a convenient tool to monitor the induced expression of the protein of interest when specific antibody is unavailable. To this end, the pEGSH vector has been engineered to provide a convenient means to detect both RNA and protein expression. For RNA detection, the T3 promoter has been positioned downstream of the MCS in the antisense orientation relative to the inducible expression cassette (Figure 2B ); labeled antisense RNA can be readily produced in vitro to use as a probe in Northern or dot blots, in situ hybridization, or RNase protection (data not shown).

To directly detect protein expression without the need for protein-specific antibody, the eight amino acid FLAG tag is positioned for C-terminal fusion to the protein of interest. Therefore, the induction of FLAG fusion proteins can be monitored by Western blotting or other immunodetection methods using the a-FLAG M2 antibody (data not shown).

Conclusions

The Complete Control system has several advantages over other inducible systems. The inducer exhibits no pleiotropic effects on cellular physiology and the inducers lipophilic nature and short in vivo half-life ensure that it rapidly penetrates and clears all tissues, as well as exhibits dose-dependent control of gene expression. In addition, the ecdysone receptor and its DNA recognition element are genetically altered so there is no cross-talk between the system and endogenous pathways. The uninduced basal activity of the system is extremely low and can be induced to over three orders of magnitude. Finally, the systems pERV3 and pEGSH vectors achieve more consistent expression of both receptors in a wide range of cell types, provide convenient means to monitor gene expression
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