Pdyn-ires Cre, Avp-ires-Cre, Crh-ires-Cre, Trh-ires-Cre and Pacap-ires-Cre mice were generated using recombineering techniques as previously described13 (link),21 (link). Briefly, a selection cassette containing an internal ribosomal entry sequence linked to Cre-recombinase and an Frt-flanked kanamycin resistance gene was targeted just downstream of the stop codon of the Prodynorphin, Arginine vasopressin, Corticotropin releasing hormone, Thyrotropin releasing hormone or Adenylate cyclase activating peptide 1 gene, respectively, in a bacterial artificial chromosome, so that Cre recombinase expression was driven by the endogenous genes. A targeting plasmid containing the Cre-containing selection cassette and 4 kb genomic sequence upstream and downstream of the Prodynorphin, Arginine vasopressin, Corticotropin releasing hormone, Thyrotropin releasing hormone or Adenylate cyclase activating peptide 1 stop codon, respectively was isolated and used for embryonic stem cell targeting. Correctly targeted clones were identified by long range PCR and injected into blastocysts. Chimeric animals generated from blastocyst implantation were then bred for germline transmission of the altered Prodynorphin, Arginine vasopressin, Corticotropin releasing hormone, Thyrotropin releasing hormone or Adenylate cyclase activating peptide 1-allele, respectively. Flp-deleter mice were then used to remove the neomycin selection cassette.
Generation of an enhanced Cre-dependent GFP reporter mice (R26-loxSTOPlox-L10-GFP) were generated using recombineering techniques as previously described20 (link). A transgene containing a lox-flanked transcriptional blocking cassette followed by eGFP fused to the L10-ribosomal subunit31 (link) was placed under the control of a CMV-enhancer/chicken beta-actin promoter and targeted to the Rosa26 locus using standard techniques32 (link). Correctly targeted blastocysts were identified by long range PCR and confirmed by southern blotting and injected into blastocysts.
Generation of an enhanced Cre-dependent GFP reporter mice (R26-loxSTOPlox-L10-GFP) were generated using recombineering techniques as previously described20 (link). A transgene containing a lox-flanked transcriptional blocking cassette followed by eGFP fused to the L10-ribosomal subunit31 (link) was placed under the control of a CMV-enhancer/chicken beta-actin promoter and targeted to the Rosa26 locus using standard techniques32 (link). Correctly targeted blastocysts were identified by long range PCR and confirmed by southern blotting and injected into blastocysts.
