Multi-transgenic minipig models exhibiting potential for hepatic insulin resistance and pancreatic apoptosis

Abstract

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.

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“…Altering insulin resistance and insulin secretion-related gene expression will make the diabetes model more closely mimic the pathology of diabetes (Association 2009). The specific mechanisms of the three genes addressed in our work and the associated model have been discussed in previous reports (Kong et al 2016;Kong et al 2015). As is known, the representative morphological change in pancreatic islets of Langerhans in T2DM is intracellular and extracellular amyloid deposition (Costes et al 2013;Hull et al 2013;O'Brien et al 1993).…”

“…In this work, we prepared dual-transgenic mice by constructing a pancreatic insulin promoter-mediated hIAPP gene and a CHOP gene overexpression vector in which the two genes were linked via Furin-2A (F-2A). The vector used to produce triple-transgenic mice was generated by overexpressing 11βHSD-1 in liver and overexpressing hIAPP and CHOP in pancreas, thereby making possible a one-step transection operation (Kong et al 2016;Kong et al 2015;Xia et al 2014) in which the hIAPP and CHOP genes were also linked by F-2A. Thus, both of these recombinant vectors can be used to transform organisms with two or three genes (Deng et al 2012).…”

“…pcDNA3.1-PapoE-11βHSD1-PIP-CHOP-F2A-hIAPP is a tissue-specific polycistronic system in which 11β-HSD1 is driven by the liver-specific PapoE (porcine apoE promoter) (Xia et al 2014) and hIAPP and CHOP are linked to the F-2A peptide, which is driven by the pancreasspecific PIP (Kong et al 2016). The PapoE (porcine apolipoprotein E promoter) sequence (Xia et al 2014), the PIP (porcine insulin promoter) sequence (Kong et al 2016), the 11β-HSD1 gene sequence (GenBank:NM_214248.1), CHOP gene sequence (GenBank: NM_007837.3) and the hIAPP gene sequence (GenBank: NM_000415.2) are stored in the National Center for Biotechnology Information (NCBI) database. The vectors pGL3-PIP-CHOP, pGL3-PIP-hIAPP-F2A-CHOP and pcDNA3.1-PapoE-11βHSD1-PIP-CHOP-F2A-hIAPP were synthesized by Generay Biotech Co. Ltd. (Shanghai, China).…”

“…The triglyceride (TG) level in the Tg animals was slightly increased, indicating increased serum lipid content. This was probably due to the increased liver lipid deposition (TG) mass, which was transported through the plasma circulation in Tg individuals (11β-HSD1 can enhance hepatic lipid deposition (Kong et al 2016;Masuzaki & Flier 2004;Paterson et al 2004)). In addition, under non-inducing treatment (Control Diet), Tg animals exhibited high serum glucose accompanied by low insulin and C peptide levels, indicating a consistent Tg insulin secretory defect trend.…”

“…Because it is difficult for these mouse models to comprehensively describe the characteristics of a disease to which susceptibility is polygenic, methods for obtaining more appropriate genetically modified disease models are an important research priority. During our work in transgenic disease model construction, we conceived the idea of combining the functional genes involved in insulin secretion defects and peripheral insulin resistance together at the genetic level (Kong et al 2016;Kong et al 2015;Lee & Cox 2011). Although there is no direct evidence in the literature concerning the interaction of the three aforementioned genes in diabetes pathogenesis, they are all important genes that can lead to diabetes (Matveyenko & Butler 2006;Oyadomari & Mori 2004;Pereira et al 2012).…”