| Over the past decades, animal models, particularly genetically modified mousemodels, have revealed many key genes and pathways that are critical inunderstanding human diseases, ontogenesis, and regeneration. Animal models havecrucial roles in biology and biomedical research. However, compared with humanbeings, mouse and other rodent animal models have extensive differences in organsize, physical structure, development cycle, and lifespan,which cannot truly mimicthe development processes of human tissues and organs. Most of the establishedmouse models can only simulate parts of human disease symptoms, and cannotreflect the disease development process. Moreover, mouse models generated forstudies on neurodegeneration, immune system disorders, and metabolic diseasesdemonstrate symptoms quite difference from those of human diseases. Large animalmodels are more similar to human beings in terms of organ size, structure, andphysiological and biochemical traits. Currently, porcine models are more suitable torecapitulate human diseases. These models are potential organ sources forxenotransplantation because of their short reproductive cycle and highreproducibility. Moreover, pigs can be raised easily and genetic modification inporcine is relatively mature, and has no ethical concerns compared with otherprimates. In addition to the roles of porcine in biological and biomedical research,they are highly important agricultural economic animals. Therefore, the geneticengineering of pigs for breeding with enhanced safety, excellent quality, diseaseresistance, and high yield is a topic of great interest in the field.However, porcine embryonic stem cells or induced pluripotent stem cells withgermline chimeric ability have not been established. Genetically modified porcinemodels are mainly generated by nuclear transfer and gene modification of somatic cells, which are achieved by specific mutation (gene targeting) in the loci of interestor random integration of the exogenous genes of interest into the genome. In recentyears, specific mutation in pig genome research has been greatly promoted by newlyemerging gene targeting technologies, such as zinc-finger nucleases, transcriptionactivator-like effector nucleases (TALENs), and RNA-guided Cas9. The randomintegration of genes, which are ectopically overexpressing foreign genes, isnecessary in producing transgenic pigs. However, the unpredictable expression ofexogenous genes and unstable phenotypes caused by position effect, copy number,and promoter methylation at random integration sites restrict the study andapplication of genetically modified pigs.Rosa26, a non-coding gene located on chromosome6of mice, was identified byrandom retroviral gene trap in mouse embryonic stem cells by Friedrich and Soriano.Rosa26is ubiquitously expressed in all types of embryonic and adult cells andtissues. The targeted modification of Rosa26results in general or conditionalexpression of transgenes, including reporters (EGFP, lacZ, and Luciferase),site-specific recombinases, transcription factors, and noncoding RNAs. Thismodification results in the establishment of over a hundred knock-in lines, which arewidely used in studies on gene function, disease mechanisms, stem cells,regenerative therapy, and RANi. ROSA26has been identified and targeted in mouse,rat, and human embryonic stem cells but not in large animals.We found a highly conserved promoter region of the ROSA26locus by multiplesequence alignment of mouse, rat, and human ROSA26sequences. We searched theporcine database using the conserved sequence as a template. A highly conservedregion with sequence similarity>85%located on pig chromosome13was observedand referred to as porcine ROSA26(pROSA26). We also performed3’RACE,RT-PCR, and quantitative PCR to demonstrate pROSA26expression in a widevariety of adult tissues. We also constructed and transiently transfected a pROSA26promoter-driven red fluorescent protein gene into various cell lines. At48hpost-transfection, high expression of red fluorescent protein was detected in all the cell lines.Thus, we applied TALEN-mediated gene targeting technology and targeted aCre recombinase-reporter gene into pROSA26with high efficiency. We generatedpROSA26gene-targeting pigs using somatic nuclear transfer, which could provide avaluable tool for studying the differentiation and regeneration of stem cells duringpig development. The generated gene-targeting pig could be used as a model toreveal the mechanism of human diseases and provide expertise in stem cell therapy.We examined the capability of pROSA26to derive foreign gene expression in vivoby transiently transfecting the Cre gene using this model. We also targeted the Cregene into a cardiac progenitor marker ISL1gene to study the development of ISL+cardiac progenitor cell in vivo.We introduced heterotypic loxP sites into pROSA26to enable the replacementof the EGFP cassette with a red fluorescent protein tdTomato gene using arecombinase mediated cassette exchange (RMCE). Thus, a large animal model forgene exchange was constructed. This model could be used to produce transgenic pigsstably overexpressing any gene of interest targeting pROSA26by RMCE. The use ofRMCE without any drug-resistant gene could eliminate the hidden problems ofbiological safety and food safety in genetically modified pigs.In this study, we first identified and characterized the porcine ROSA26locus.We generated Cre-inducible reporter pigs for cell lineage tracing usingTALEN-mediated homologous recombination and somatic cell nuclear transfer. WithRMCE, any gene of interest could be targeted into the pROSA26locus andubiquitously expressed in pig. This study successfully solve problems caused byconventional random integration of genes and overcome the hidden troubles of theresistance gene carried by the genetically modified pigs, which have a greatimprovement on the research and application of pigs in biomedical and agricultural. |
