Rosa26 locus supports tissue-specific promoter driving transgene expression specifically in pig

• Published in: PloS one Vol. 9; no. 9; p. e107945
• Main Authors: Kong, Qingran, Hai, Tang, Ma, Jing, Huang, Tianqing, Jiang, Dandan, Xie, Bingteng, Wu, Meiling, Wang, Jiaqiang, Song, Yuran, Wang, Ying, He, Yilong, Sun, Jialu, Hu, Kui, Guo, Runfa, Wang, Liu, Zhou, Qi, Mu, Yanshuang, Liu, Zhonghua
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.09.2014; Public Library of Science (PLoS)
• Subjects: Agricultural research; Agriculture; Animal genetic engineering; Animals; Animals, Genetically Modified; Base Sequence; Biology; Biology and Life Sciences; Biomedical materials; Biotechnology; Cells, Cultured; CRISPR; Deoxyribonucleic acid; DNA; DNA methylation; Elongation; Follistatin; Follistatin gene; Gene Expression; Gene targeting; Gene therapy; Genes, Reporter; Genetic Loci; Genetic modification; Genetically modified animals; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Hogs; Homologous recombination; Homology; Laboratories; Life sciences; Loci; Methylation; Molecular Sequence Data; Muscle, Skeletal - metabolism; Muscles; Mutation; Myostatin; Neurosciences; Organ Specificity; Phenotypes; Promoter Regions, Genetic; Reporter gene; RNA, Untranslated - genetics; Rodents; Stem cells; Suidae; Sus scrofa - genetics; Swine; Swine, Miniature; Transcriptional Activation; Transgenes; Zoology; Beijing China; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0107945

Genetically modified pigs have become a popular model system in fundamental research, agricultural and biomedical applications. However, random integration often result in unstable expression of transgene and unpredictable phenotypes. The Rosa26 locus has been widely used to produce genetic modified animals with high and consistent expressing of transgene in mouse, human and rat, as it can be targeted efficiently and is not subject to gene-silencing effects. Recently, the first case of reporter gene targeting pigs in porcine Rosa26 (pRosa26) locus was reported. In the study, full sequence of pRosa26 locus was further characterized, and the pRosa26 promoter (pR26) was cloned and we evidenced that the new porcine endogenous promoter is suitable for driving transgene expression in a high and stable manner by avoiding DNA methylation. Furthermore, elongation factor 1a promoter (EF1a) -driven GFP reporter and Myostatin promoter (MyoP)-driven Follistatin (Fst) were successfully targeted into the pRosa26 locusby traditional homologous recombination (HR) strategy. EF1a showed high activity and hypomethylation at the locus. And, muscle-specific promoter MyoP was activated strictly in muscle of the pRosa26 targeted pigs, indicating Rosa26 locus supports tissue-specific promoter driving transgene expression in its own manner. The study provided further demonstration on biomedical and agricultural applications of porcine Rosa26 promoter and locus.