Figure 1. Generation of Vert-X (fr. green, 10) mice: Targeting of an IRES-eGFP reporter into the il10 locus.
(a) Maps of the il10 locus (top), the targeting construct (middle), and the mutated il10 locus (bottom). A genomic 6.8 kb EcoRI fragment of the wildtype C57BL/6 locus (top), isolated by recombineering techniques from a BAC clone, was mutated by: (i) the introduction of a loxP-flanked (filled triangles) neomycin cassette (Neo), an internal ribosomal entry site (IRES) element, enhanced green fluorescent protein (EGFP), and a polyadenylation signal (pA), all cloned into a HindIII site between the 5th exon and the endogenous pA of il10; and (ii) the 5′ addition of an HSV-derived thymidine kinase (HSV-tk) cassette. After verification (by PCR and Southern blotting) of successful targeting into C57BL/6 embryonic stem cells, positive clones were injected into blastocysts of C57BL/6 albino females. The floxed Neo was deleted by Cre-mediated recombination using C57BL/6 Zp3 Cre deletor mice (Jackson), generating the targeted il10 locus depicted at bottom. Numbered boxes represent exons. (b) Southern blot analysis of DNA from tail biopsies of the progeny of heterozygous breeding. The novel NsiI site introduced by the targeting construct results in a 3 kb fragment in the correctly targeted allele, in place of the 10 kb fragment of the wildtype allele. +, wildtype; Vert-X, targeted allele.