Kong Siyuan, Ruan Jinxue, Xin Leilei, Fan Junhua, Xia Jihan, Liu Zhiguo, Mu Yulian, Yang Shulin, Li Kui
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China.
Mol Med Rep. 2016 Jan;13(1):669-80. doi: 10.3892/mmr.2015.4582. Epub 2015 Nov 19.
There are currently no multi‑transgenic minipig models of diabetes for the regulation of multiple genes involved in its pathogenesis. The foot and mouth disease virus 2A (F2A)‑mediated polycistronic system possesses several advantages, and the present study developed a novel multi‑transgenic minipig model associated with diabetes using this system. The tissue‑specific polycistronic system used in the present study consisted of two expression cassettes, separated by an insulator: (i) 11‑β‑hydroxysteroid dehydrogenase 1 (11β‑HSD1), driven by the porcine liver‑specific apolipoprotein E promoter; (ii) human islet amyloid polypeptide (hIAPP) and C/EBP homologous protein (CHOP), linked to the furin digested site and F‑2A, driven by the porcine pancreas‑specific insulin promoter. In the present study, porcine fetal fibroblasts were transfected with this vector. Following somatic cell nuclear transfer using 10 cell clones and the transplantation of 1,459 embryos in total, three Landrace x Yorkshire surrogates became pregnant and delivered three Wuzhishan piglets. Genomic polymerase chain reaction (PCR) demonstrated that the piglets were multi‑transgenic. Reverse transcription‑quantitative PCR confirmed that 11β‑HSD1 transcription was upregulated in the targeted liver. Similarly, hIAPP and CHOP were expressed at high levels, compared with the control (P<0.05 and P<0.01) in the pancreas, consistent with the western blotting and immunohistochemistry results. The primary results also showed that overexpression of 11β‑HSD1 in the liver increased the liver fat lipid parameters; and the levels of hIAPP and CHOP in the pancreatic islet cells, leading to delayed β‑cell development and apoptosis. This novel tissue‑specific polycistronic system offers a promising starting point for efficiently mimicking multigenic metabolic disease.
目前尚无用于调控糖尿病发病机制中多个相关基因的多转基因小型猪模型。口蹄疫病毒2A（F2A）介导的多顺反子系统具有多种优势，本研究利用该系统构建了一种新型的与糖尿病相关的多转基因小型猪模型。本研究中使用的组织特异性多顺反子系统由两个表达盒组成，中间由一个绝缘子隔开：（i）由猪肝特异性载脂蛋白E启动子驱动的11-β-羟基类固醇脱氢酶1（11β-HSD1）；（ii）由猪胰腺特异性胰岛素启动子驱动的人胰岛淀粉样多肽（hIAPP）和C/EBP同源蛋白（CHOP），它们与弗林蛋白酶切割位点和F-2A相连。在本研究中，用该载体转染猪胎儿成纤维细胞。使用10个细胞克隆进行体细胞核移植并总共移植1459个胚胎后，三头长白猪与约克夏猪的代孕母猪怀孕并产下三头五指山仔猪。基因组聚合酶链反应（PCR）表明这些仔猪是多转基因的。逆转录定量PCR证实，在靶向肝脏中11β-HSD1转录上调。同样，与对照组相比，胰腺中hIAPP和CHOP的表达水平较高（P<0.05和P<0.01），这与蛋白质免疫印迹和免疫组织化学结果一致。初步结果还表明，肝脏中11β-HSD1的过表达增加了肝脏脂质参数；胰岛细胞中hIAPP和CHOP的水平升高，导致β细胞发育延迟和凋亡。这种新型的组织特异性多顺反子系统为有效模拟多基因代谢疾病提供了一个有前景的起点。
