Abstract
ABSTRACTThe Wistar Kyoto (WKY) rat and the spontaneously hypertensive (SHR) rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN) and metabolic syndrome, respectively. Novel transgenic (Tg) strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP) under the rat elongation factor 1 alpha (EF1a) promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.
Highlights
The rat has long been an important physiological model of complex human disease (Aitman et al, 2008; Iannaccone and Jacob, 2009)
With the advent of gene targeting technologies that are suited to use in rats, including zinc finger nucleases (ZFNs) (Geurts et al, 2009), transcription activator-like effector nucleases (TALENs) (Tesson et al, 2011), clustered regularly interspaced short palindromic repeats (CRISPRs) (Shen et al, 2013) and Sleeping Beauty (SB) transposon system (Ivics et al, 2009; Kitada et al, 2007; Mátés, 2011), increasing numbers of new transgenic (Tg), knockout and knock-in rat models have been created to elucidate the functional basis of disease phenotypes (Jacob et al, 2010)
One high-green fluorescent protein (GFP)-expresser F0 per strain was used to derive each transgenic (Tg) rat line. Both lines showed normal growth, were able to reproduce and germline transmit the transgene, and after more than five generations, GFP expression was maintained without any sign of transgene silencing
Summary
The rat has long been an important physiological model of complex human disease (Aitman et al, 2008; Iannaccone and Jacob, 2009). Received 25 November 2015; Accepted 13 January 2016 of the human hypertension and metabolic syndrome as well as a large number of other pathophysiological phenotypes including cardiac hypertrophy and failure, insulin resistance and defects in lipid metabolism (Aitman et al, 1999, 1997; Lepretre et al, 2004; Pravenec et al, 2008; Yamori and Okamoto, 1974) Both inbred strains are well characterised, with 199 and 197 quantitative trait loci (QTLs) mapped, respectively, in WKY and SHR (Shimoyama et al, 2015), and whole genome sequences of both strains publically available (Aitman et al, 2008; Atanur et al, 2010, 2013; Hubner et al, 2005; Shimoyama et al, 2015). With the advent of gene targeting technologies that are suited to use in rats, including zinc finger nucleases (ZFNs) (Geurts et al, 2009), transcription activator-like effector nucleases (TALENs) (Tesson et al, 2011), clustered regularly interspaced short palindromic repeats (CRISPRs) (Shen et al, 2013) and Sleeping Beauty (SB) transposon system (Ivics et al, 2009; Kitada et al, 2007; Mátés, 2011), increasing numbers of new transgenic (Tg), knockout and knock-in rat models have been created to elucidate the functional basis of disease phenotypes (Jacob et al, 2010)
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