Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.

Clinical assessment after human adipose stem cell transplantation into dogs

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.

Mineralized deposits in the uterus of a pig without pregnancy loss

Herein, we describe a case of uterine calcification in the uterus of a pig without pregnancy loss. The recipient underwent cloned embryo transfer and Cesarean section for safe delivery of cloned piglets. During the Cesarean section, 4 white, star-like, (2 × 2 × 2) cm, calcified structures were found within the endometrial cavity. Despite dystrophic calcification around the placenta, healthy cloned piglets were produced successfully. To our knowledge, this is the first reported case of dystrophic calcification occurring within the uterus in a pregnant pig.

Immunohistochemical localization of glucose transporter 1 and 3 in the scrotal and abdominal testes of a dog / 한국실험동물학회지

Glucose is essential for testicular function; the uptake of carbohydrate-derived glucose by cells is mediated by glucose transporters (GLUTs). In the present study, we investigated the activity of GLUT1 and GLUT3, the two main isoforms of GLUTs found in testes, in the left scrotal and right abdominal testes of a German Shepherd dog. Immunohistochemical analysis showed that GLUT1 immunoreactivity was absent in the scrotal and abdominal testes. In contrast, weak to moderate GLUT3 immunoreactivity was observed in mature spermatocytes as well as spermatids in the scrotal testis. In the abdominal testis, relatively strong GLUT3 immunoreactivity was detected in Leydig cells only and was absent in mature spermatocytes and spermatids. GLUT3 immunoreactivity was significantly decreased in the tubular region of abdominal testis and significantly increased in the extra-tubular (interstitial) region of abdominal testis compared to observations in the each region of scrotal testis, respectively. These results suggest that GLUT3 is the major glucose transporter in the testes and that abdominal testes may increase the uptake of glucose into interstitial areas, leading to an increased risk of developing cancer.

Reproductive ability of a cloned male detector dog and behavioral traits of its offspring

In 2007, seven detector dogs were produced by somatic cell nuclear transfer using one nuclear donor dog, then trained and certified as excellent detector dogs, similar to their donor. In 2011, we crossed a cloned male and normal female by natural breeding and produced ten offspring. In this study, we investigated the puppies' temperaments, which we later compared with those of the cloned parent male. The results show that the cloned male had normal reproductive abilities and produced healthy offspring. All puppies completed narcotic detector dog training with a success rate for selection of 60%. Although the litter of cloned males was small in this study, a cloned male dog bred by natural mating produced puppies that later successfully completed the training course for drug detection. In conclusion, cloning an elite dog with superior genetic factors and breeding of the cloned dog was found to be a useful method to efficiently procure detector dogs.

Learning, memory and exploratory similarities in genetically identical cloned dogs

Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals.

Differential expression of estrogen receptor α and progesterone receptor in the normal and cryptorchid testis of a dog / 한국실험동물학회지

Descending of the testes is an important process for spermatogenesis and cryptorchidism is one of the most relevant genital defects in dogs. In a previous study, we observed abnormal morphology and proliferation of Sertoli cells in a cryptorchid testis. In the present study, we investigated the expression of estrogen and progesterone receptors in the normal and cryptorchid testis of a dog. Elective orchidectomy was performed on the dog's abdominal right testis (undescended, cryptorchid) and scrotal left testis (descended, normal). In the normal testis, estrogen receptor α immunoreactivity was detected in Leydig cells alone, while estrogen receptor α immunoreactivity in the cryptorchid testis was significantly prominent in the Sertoli cells as well. In addition, progesterone receptor immunoreactivity in the control testis was detected in the spermatids, but was not detected in the cryptorchid testis. This result suggests that unilateral cryptorchidism causes increases of estrogen receptor α expression in Sertoli cells.

Unilateral cryptorchidism induces morphological changes of testes and hyperplasia of Sertoli cells in a dog / 한국실험동물학회지

Cryptorchidism is one of the most common genital defects in dogs. This study investigated the effects of abdominal cryptorchidism on morphology, cell proliferation, and Sertoli cell condition in a dog with spontaneous unilateral cryptorchidism. Elective orchidectomy was performed on the abdominal right testis and the scrotal left testis. Significant reductions in numbers of spermatogonia, spermatocytes, and spermatids were observed in hematoxylin and eosin stained sections of the cryptorchid testis. The size of the epididymal duct was smaller than that of the control testis. Based on Ki67 immunohistochemistry, the proliferative activity of spermatogonia and spermatocytes was significantly decreased in the cryptorchid testis. However, proliferative activity was increased in the epididymal duct. Based on GATA-4 immunohistochemistry, Sertoli cells were relatively resistant to cryptorchidism, and the proliferative activity of Sertoli cells was markedly increased in the cryptorchid testis than in the control testis. These results suggest that spontaneous unilateral cryptorchidism causes morphological defects in spermatogonia and spermatocytes in the testis and changes the size of the efferent ductule of the epididymis. In addition, spontaneous unilateral cryptorchidism increases proliferative activity of Sertoli cells, which may be a predisposing factor for Sertoli cell cancer in cryptorchid testes.

Post-mortem re-cloning of a transgenic red fluorescent protein dog

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.