Production Of A Reporter Transgenic Pig For Monitoring Cre Recombinase Activity

The Cre-LoxP system has been developed as a powerful tool for manipulating DNA both in vitro and in vivo. Though standard gene knockouts have been highly informative, early embryonic lethality or complex phenotypes often obscure the roles of genes studied during later stages of development or in specific tissues. Conditional gene targeting provides a means to circumvent certain limitations of conventional gene targeting. the pig is thought to be the most suiTab non-human source of organs for xenotransplantation, and it is becoming more widely used as a model of human disease. Effective use of conditional Cre recombinase-LoxP gene modification requires Cre-expressing porcine strains with defined patterns of expression, for example by the use of a tissue-specific promoter. Such pigs would be a powerful tool for studying human gene expression. In order to monitor recombinant Cre expression, it is important to create reporter strains.At first, we constructed a vector, in this vector, the EGFP gene is regulated by a CMV promoter, but its expression is inhibited by a LoxP-flanked intervening cassette containing the neo gene, followed by transcriptional termination sequences. The final vector was named pICE-STOP. To generate transgenic cells, porcine fetal fibroblasts were seeded in a 60mm dish at a density of 2×105 cells per dish on the day before transfection. Cells were cultured in DMEM supplemented with 15% fetal bovine serum and incubated at 39 oC. For the production of stably trasfected cell lines, the pICE-STOP plasmids were digested with NheI. On the following day, the pICE-STOP fragment was incorporated into Lipofectamine 2000 ? reagent, after cells were cultured in selection medium containing 300μg /ml of G418 antibiotic for an additional 7 days. The surviving cell colonies were picked and propagated and the cells were then frozen to be used as the donor cells.In order to examine if the gene have intergrated into genome, the genomic DNA of clones was used for PCR amplification and intergratd sites analyzed by high-efficiency thermal asymmetric interlaced PCR.To examine excision-activated EGFP expression in these cells, the clones were individually assayed for expression of EGFP following infection with Ad-Cre at an MOI of 800 for 72 h. Images were collected on a laser scanning confocal microscope .At 72 h after Ad-Cre transfection, levels of EGFP mRNA were measured by RT–PCR using theprimers selected for detection of the EGFP sequence. FACS analysis was also used to characterize the expression of EGFP.A positive cell clone was used to construct a transgenic pig by nuclear transfer (NT). To determine whether Cre can regulate the expression of EGFP, transgenic pig cells were isolated from tails that were infected with recombinant Cre adenovirus.Two days after rAd infection, EGFP gene expression was assessed with a laser scanning confocal microscope. Total RNA was purified and RT-PCR was performed.We generate transgenic reporter pigs, which can be used to monitor Cre-mediated excision. The EGFP gene is expressed only after Cre-mediated excision of LoxP-flanked stop sequences. We expect that this reporter strain could facilitate the in vivo monitoring of Cre-mediated excision events in a variety of experimental contexts.