Garcia Diaz Ana Isabel, Moyon Ben, Coan Philip M, Alfazema Neza, Venda Lara, Woollard Kevin, Aitman Tim
Division of Immunology and Inflammation, Imperial College London, London W2 1PG, UK MRC Clinical Sciences Centre and Department of Medicine, Imperial College London, London W12 0NN, UK.
Embryonic Stem Cell and Transgenics Facility, MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, UK.
Dis Model Mech. 2016 Apr;9(4):463-71. doi: 10.1242/dmm.024208. Epub 2016 Jan 14.
The 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 preliminaryin vitroandin vivoimaging 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.
Wistar Kyoto（WKY）大鼠和自发性高血压（SHR）大鼠近交系分别是人类新月体性肾小球肾炎（CRGN）和代谢综合征的成熟模型。新型转基因（Tg）品系为成熟的大鼠模型增加了研究机会并提高了科学价值。我们利用睡美人转座子种系转基因技术创建了两个新型Tg品系，在WKY和SHR遗传背景下，在大鼠延伸因子1α（EF1a）启动子的控制下普遍表达绿色荧光蛋白（GFP）。睡美人系统具有很高的转基因效率；胚胎显微注射后出生的新大鼠中有75%是转基因阳性。通过连接介导的PCR，我们定位了基因组整合位点，证实没有外显子破坏，并确定了新的WKY-GFP和SHR-GFP Tg系中转基因的单拷贝或低拷贝数。我们报告了两个品系的胚胎、组织和器官中GFP的明亮表达，并展示了初步的体外和体内成像数据，这些数据证明了新的GFP表达品系在CRGN、代谢综合征以及在过去四十年中对这些品系进行了广泛研究的其他性状的过继转移、移植和命运图谱研究中的实用性。
