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. 2009 Mar 11;37(7):e55. doi: 10.1093/nar/gkp112

Figure 3.

Figure 3.

Generation of MultiSite Gateway-compatible ROSA26-targeted pCAGG-promoter-based expression alleles. (A) Generation of MultiSite ROSA26 targeting vectors using the 5′-pCAGG-loxP flanked β-geo-3xpA (STOP) cassette, middle-cDNA and 3′-IRES-eGFP reporter pEntry clones. (B) In the conditional knock-in alleles before Cre-mediated excision the pCAGG promoter drives the expression of the β-geo (β-galactosidase-neomycin phosphotransferase fusion gene) cassette (arrows) but not downstream cDNA-IRES-eGFP mRNA expression. Shown are the expected fragment lengths using KpnI/EcoRI digests and the 5′ and 3′ as well as internal eGFP probes used to confirm both sense and anti-sense orientation pCAGG-based single-integration transgenes to the ROSA26 locus. (C) Following Cre-mediated excision of the floxed β-geo cassette, the pCAGG promoter drives cDNA-IRES-eGFP transgene expression in either a sense or anti-sense orientation relative to the sense ROSA26 promoter.