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Figure 3. Cellular tool to modulate gene activity of inactivated or overexpressed genes. A bidirectional RMCE (recombinase-mediated cassette exchange) strategy is used to modulate gene activity by sequence specific recombination of genomic BAC-clone inserts or gene-specific shRNA coding inserts. The selection cassette coded by the targeting vector is integrated randomly into the genome and selected by neomycin resistance to monclonality. The vector pBACe3.6 of the RP11-BAC library encodes the loxP- and loxP511-sites, flanking the genomic insert. The vector pSUPERdL ("dL" for "double Lox") was obtained from the pSUPER vector, modified with the two respective lox-sites. By cotransfection of a Cre-expression plasmid (pCMXhCre) and either a pBACe3.6-clone or an RNAi-plasmid pSUPERdL the single-copy transgen between the loxP511- and loxP-sites is exchanged, selectable by an HSV-Tk counterselection with ganciclovir (GCV).
