Abstract
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.
Highlights
Modified pigs hold great promise in the fields of fundamental research, agriculture and biomedicine [1]
To get the ncRNA of porcine Rosa26 (pRosa26), we designed primers aligned within the predicted exon 1 and ESTs locating to the region to perform RT-PCR. 2 exons flanking 1 intron were amplified with the primers based on the EST sequence of EW546160, and the pRosa26 ncRNA was transcribed in the opposite strand of one ThumpD3 gene (Figure S2). 39 and 59 RACE were performed and a 646 bp transcript with termination signal was identified, suggesting at least we got the full-length of one transcript variant of pRosa26 (FigureS3 and Figure 1A)
This study describes the identification and characterization of the porcine Rosa26 promoter and locus, and demonstrates the activities of ubiquitous and tissue-specific promoters at the locus, providing an important advancement for genetical modification inpig
Summary
Modified pigs hold great promise in the fields of fundamental research, agriculture and biomedicine [1]. Gene targeting technologies permit the insertion of exogenous constructs into defined genomic sites Available approaches, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system, are capable of inducing double-stranded breaks (DSBs) to enhance homologous recombination for producing gene-knockin pigs [5,6,7,8]. Off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect and cytotoxicity happens due to the cell’s emergency response to DSBs [9,10,11,12,13,14,15] These remain concerns for the clinical use of nucleasebased approaches. Due to unavailability of the germline-competent stem cells, gene-knockin pig by traditional HR is not obtained yet
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