We have previously generated a transgenic mouse strain (LSL-TRICA) containing a

We have previously generated a transgenic mouse strain (LSL-TRICA) containing a Cre-inducible constitutively active TGF type I receptor (Bartholin L. kb when the STOP signal is removed after Cre-mediated recombination. We validated excision in several compartments, including pancreas, liver, T lymphocytes and embryos using different Cre expressing transgenic mouse strains. This represents a simple and efficient way of monitoring the tissue specific recombination of the LSL-TRICA allele. step for transgene expression. The possibility to monitor this genomic recombination represents a convenient, reliable and very sensitive strategy to validate Cre-mediated recombination in the compartment of interest. We generated females with the three following genotypes: wild type (+/+, no TRICA), heterozygous (lox/+, one TRICA allele) and homozygous (lox/lox, two TRICA alleles). Genotyping PCR experiments were performed on genomic DNA extracted from different organs samples using the three set of primers. We previously reported (Bartholin in a whole embryo expressing the Cre recombinase. We next attempted to determine whether organ-specific recombination could be detected. To that end we tested specific recombination in three different compartments: pancreas, liver and T lymphocytes. LSL-TRICA mice were crossed with Pdx1-Cre mice, which express the Cre recombinase during early embryogenesis in pancreatic cells from all lineages (endocrine, acinar, centroacinar and ductal cells) (Gu em et al. /em , 2002). LSL-TRICA mice were also crossed with Alb-Cre mice (Postic em et al. /em , 1999) (expression of Cre recombinase in the liver under the control of the AZD2171 enzyme inhibitor albumin promoter) and CD4-Cre mice (Lee em et al. /em , 2001) (expression of Cre recombinase in T lymphocytes under the CD4 promoter). Recombination in the progeny obtained from these crosses and bearing both Cre and LSL-TRICA transgenes was then analyzed by PCR (Physique 2C). As expected, the 0.35 kb AZD2171 enzyme inhibitor recombined band was observed only in organs that express Cre recombinase, e.g. in the pancreas of Pdx1-Cre; LSL-TRICA, in the liver of Alb-Cre; LSL-TRICA, and in the T lymphocytes of CD4-Cre; LSL-TRICA. In each case PCR performed from tails snips or ear punches revealed the presence of the 1.93 kb band attesting to the presence of the non recombined allele. This represents a simple and efficient way of monitoring the recombination status of the LSL-TRICA allele. Materials and Methods Mice We Rabbit Polyclonal to SFRS7 generated LSL-TRICA transgenic animals (Bartholin em et al. /em , 2008) by inserting at the Hprt locus of the Lox-STOP-Lox cassette situated upstream of constitutively active TGF type I mutant receptor due to three missense mutations: T204D that constitutively activates the TRI kinase (Wieser em et al. /em , 1995) and L193A/P194A that prevent binding of the TRI inhibitor, FKBP12 (Charng em et al. /em , 1998). Pdx1-Cre (pancreas) (Gu em et al. /em , 2002), CD4-Cre (T lymphocytes) (Lee em et al. /em , 2001), Alb-Cre (Liver) (Postic em et al. /em , 1999) and Sox2-Cre (embryos) (Hayashi em et al. /em , 2002). Mouse strains were previously explained and published by others. All animals were dealt with in accordance with either institutional or regional guidelines. Samples preparation DNA was isolated and purified from different tissues according to the standard HotShot protocol (Truett em et al. /em , 2000) (alkaline lysis at 95C for 30 min before acidic neutralization). More precisely, for mouse pancreas preparation, pancreas was diced with a scalpel and incubated in collagenase IA 200U/ml (Sigma), Hepes 10 mM (Invitrogen), Soybean trypsin inhibitor 0.25 mg/ml (Sigma) in HBSS (Invitrogen). T lymphocytes were purified with an AutoMACS Pro magnetic cell sorter and magnetic beads (Miltenyi Biotec, Gladbach, Germany) using an anti-mouse CD4 (L3T4, BD Biosciences) as previously explained (Marie em et al. /em , 2006). Embryos were dissected out at E7.5 without yolk-sac liver samples were prepared from very small piece of liver, and did not undergo further treatment before HotShot DNA extraction procedure. PCR To visualize recombined LSL-TRICA allele (pCAG/pTRI primer set), PCR was carried out using and 2 l of neutralized supernatant per 25 l PCR reaction. The reaction combination made up of 0.2 M of each primers, 0.01 U/l Platinium Taq DNA polymerase, 0.2 mM dNTP, 1.5 mM MgCl2 (Invitrogen). The samples were in the beginning heated at 94C for 5 min followed by 35 cycles, each consisting of 30 second at 94C, 1 min 30 seconds at 58C, and 2 moments 30 s at 72C and a final extension at 72C AZD2171 enzyme inhibitor for.