Production of Transgenic Dogs Carrying Human Phosphoenolpyruvate Carboxykinase and Amyloid Precursor protein Gene by Nuclear Transfer

Abstract

Dog cloning is of immense importance to human society due to its emotional and therapeutic values. Lately, with the current burgeoning necessity of canine genetic information for reflecting human disease, transgenic dogs carrying human genes associated with a certain disease could provide a therapeutic tool and preclinical model for testing therapeutic drugs. The aim of the present study was to improve canine cloning technology by studying the relationship between different types of dog breeds as somatic cell donor on the cloning efficacy and to establish the transgenic cell lines produce the transgenic cloned dogs carrying human phosphoenolpyruvate carboxykinase and amyloid precursor protein gene, and characterize their pathophysiology. At the outset of this study, using 10 tentative nuclei donors for nuclear transfer, the effects of different types of donor cells, the meiotic stage of the recipient oocyte, the passage of the donor nuclei on the cloning efficiency and the postnatal development of resulted cloned puppies were studied. In the later part of the study, two genes implicated with the devastating human disease such as type 2 diabetes mellitus or Alzheimers diseases were chosen as the candidate genes, which were transfected into dog fibroblasts and established the stable cell line for the nuclear donor. The breeds providing the donor nuclei for nuclear transfer were categorized into four groups according to their body weight. Reproductive parameters were evaluated in transferring reconstructed embryos from each breed into the surrogates. The number of live pups delivered in the ultra large group was significantly higher compared to the other groups (P <0.05). There was no direct correlation between the number of embryo transferred and the litter size. The highest litter size was examined in ultra-large breeds. The mean birth weight of cloned pups significantly higher in the ultra-large group and the large group compared to the other groups (P <0.05). A significantly shorter gestation period was observed in the small breed compared to others (P <0.05). There was no significant difference in the pregnancy maintenance dependent on the breed size. The meiotic maturation of the recipient oocytes and the passages of donor nuclei do not affect the cloning efficiency. However, reconstructed embryos from the immature oocytes used as recipient cytoplasts had neither attached nor implanted, on the other hand, those from the aged oocytes had maintained a full term pregnancy. Beagle was chosen as the nuclei donor in the second round of the study. For the establishment of the genetically modified cell line, fibroblasts were surgically obtained from the fetuses that are of 25 days after artificial insemination. Two different genes, phosphoenolpyruvate carboxykinase for type 2 diabetes mellitus and human amyloid precursor protein gene for Alzheimers disease, were designed and controlled by a selective promoter containing dual selection marker genes (green fluorescence gene and neomycin resistant gene). In order to establish the type 2 diabetes mellitus dog model, the transgenic cells carrying phosphoenolpyruvate carboxykinase were selected and inserted into the enucleated canine oocytes. Forty-seven reconstructed embryos were transferred into five surrogate mothers resulting in two pregnancies. Three puppies were born and confirmed genetically identical with the donor using the microsatellite analysis. Using PCR, one puppy was confirmed to carry the exogenous phosphoenolpyruvate carboxykinase gene. Relatively high expression of phosphoenolpyruvate carboxykinase gene was observed in the liver biopsy using RT-PCR. No symptoms directly reflected to type 2 diabetes mellitus were observed in the dog. To establishment for Alzheimers disease dog model, transgenic cells expressing the human amyloid precursor protein gene containing well-characterized familial Alzheimers disease mutations was selected and used for the donor nuclear. Founder transgenic male and female were produced and mated to produce the second generation. The expression of the transgene was confirmed by observing the expression of green fluorescence protein in the body as a visual transgene marker. The presence and expression of the mutated amyloid precursor protein genes were determined in the brain. One transgenic dog showed spontaneous tetanic convulsion that were examined in Alzheimers disease-like symptoms such as enlarged ventricles, atrophied hippocampus, and β-amyloid plaques in the brain. Taken together, the efficiency of cloning and fetal parameter after the embryo transfer in the canine could be improved by selecting the appropriate genotype. The transgenic dogs expressing phosphoenolpyruvate carboxykinase and mutant amyloid precursor protein gene could be produced using lipofection-mediated transgenic technique. These findings may contribute to better understand the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus and human Alzheimer's disease.