Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase

Abstract

Abstract. Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.

Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals

“…Owing to a shared environment and to similarities in physiology, disease presentation, and clinical response at least half of canine diseases are known to have human equivalents making the dog an ideal model for human disorders (Starkey et al, 2005). Although controversial (Varner, 1999;Fiester, 2005), using SCNT to generate genetically modified disease models in dogs looks promising (Jeong et al, 2012;Oh et al, 2012). However, as with other domestic animal clones (Wells, 2005), there have also been some reports of abnormalities in cloned dogs that would currently limit this use (Kim et al, 2009;Hong et al, 2011a).…”

Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases

“…We reasoned that primary canine broblasts derived from less mature donor tissues may undergo reprogramming towards pluripotency more e ciently; which has been linked to both proliferative capacity and various nuclear determinants of the undifferentiated state (48,49). Canine fetal broblasts (cFFs) reported to be permissive to somatic cell nuclear transfer-mediated reprogramming (40) were assessed for their suitability as donor cells for OSKM transcription factor mediated reprogramming. Early passage cFF cells exhibit a more rapid proliferation rate compared to early passage cAFs (Supplemental Fig.…”

“…Research Foundation (Seoul, South Korea) (40). Adult or fetal canine broblasts were maintained in highglucose DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMax, 1X non-essential amino acids and 0.1 mM 2-mercaptoethanol.…”

Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

“…Fibroblasts were isolated from dog (Canis lupus familiaris) fetuses on day 39 of gestation and from the ear skin of each pup as previously described [20]. Briefly, tissues from fetus were washed in phosphate-buffered saline (PBS), containing 1% (v/v) antibiotic-antimycotic (Life Technologies) and chopped finely using a blade.…”