• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2009.02a
• /
• pp.49-57
• /
• 2009

The cloning of canids was succeeded in 2005, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay of successful somatic cell nuclear transfer (SCNT)was due to the unique reproductive characteristics of the female dogin comparison to other domestic mammals, such as ovulation of immature canine oocyte and a requirement of 25 days for the completion of meiosis within the oviduct (Holst & Phemister, 1971). When the technology for the recovery of in vivo matured oocyte was established, the application of cloning also became possible and cloned dog offspring were obtained. This report summarizes the progress of technical procedures that are required for cloning canids and the application of this technique. The first cloned dog, Snuppy, was achieved using an in vivo-matured oocyte which was enucleated and transferred with an adult skin cell of male Afghan hound. After establishment of a criterion of well-matured oocyte for the improvement of SCNT efficiency, we obtained three cloned female Afghan hound and a toy poodle cloned from 14 year-old aged Poodle using SCNT through this factor. To date, cloned dogs appeared to be normal and those that have reached puberty have been confirmed to be fertile. Through application of canine SCNT technique, first, we demonstrated that SNCT is useful for conserving the breed of endangered animal from extinction through cloning of endangered gray wolves using inter-species SCNT and keeping the pure pedigree through the cloning of Sapsaree, a Korean natural monument. Secondly, we showed possibility of human disease model cloned dog and transgenic cloned dog production through cloning of red fluorescent protein expressing dog. Finally, SCNT can be used for the propagation of valuable genotypes for making elite seed stock and pet dog. In summary, dog cloning is a reproducible technique that offers the opportunity to preserve valuable genetics and a potential step towards the production of gene targeted transgenic cloned dogs for the study of human diseases.

• The Korean Journal of Physiology and Pharmacology
• /
• v.71 no.3
• /
• pp.351-358
• /
• 2020

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)-derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

• Animal Bioscience
• /
• v.35 no.9
• /
• pp.1360-1366
• /
• 2022

Objective: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

• Journal of Veterinary Clinics
• /
• v.23 no.4
• /
• pp.458-460
• /
• 2006

A 2-year-old female, 3.8kg weight, mixed-breed dog was presented for a 10-day history of vomiting and anorexia. The dog was diagnosed with pelvic fracture by vehicular accident at a local animal hospital before 2 months, but fracture did not reconstructed. After 2 months, the dog suffered from intermittent vomiting and anorexia. On barium contrast, we diagnosed the dog as intestinal obstruction. Consequently, we were found obstruction by strangulation and adhesion of intestine at the fracture site through the operation. At present after 18 months of surgery, general physical condition of the dog is good.

• Journal of the East Asian Society of Dietary Life
• /
• v.7 no.3
• /
• pp.289-300
• /
• 1997

Newborn dog chymosin was extracted from the stomachs of dogs of 2 weeks of age, and was purified by ion exchange chromatography. Half of the sequence was determined by amino acid sequencing and the complete sequence was deduced from a cloned chymosin cDNA Results showed that the zymogen showed 79% sequence identity with calf prochymosin and 54% identity with porcine pepsinogen A The size of the propart and location of the residue which becomes the amino-terminus in the active enzyme was the same in the prochymosins. The maximum general proteolytic activity at pH 3.2 of newborn dog chymosin was 3-4% of that of porcine pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of newborn dog chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.2 of stomach extracts of individual dogs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or amino-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and newborn dog chymosin differ in the following positions (porcine pepsin numbering, subsites in parentheses) : Ser 12 Thr(S$_4$), Leu 30 Val(S$_1$/S$_3$), His 74 Gln(S'$_2$), Val 111 Ile(S$_1$/S$_3$), Lys 220 Met(S$_4$). With regard to the low general proteolytic activity of newborn dog chymosin, the substitution Asp303 Val relative to calf chymosin may contribute to an explanation of this.

• Journal of Embryo Transfer
• /
• v.28 no.3
• /
• pp.229-235
• /
• 2013

The objective of this study was to monitor health conditions of four genetically identical somatic cells cloned Labrador retriever puppies by estimation of body weight and analysis of hematologic and serologic characteristics. Naturally ovulated oocytes and donor cells were used for somatic cell nuclear transfer (SCNT). Donor cells and enucleated oocytes were followed by electric fusion, chemical activation and surgical embryo transfer into the oviducts of surrogate females. Two recipients became pregnant; two maintained pregnancy to term, and four live puppies were delivered by Caesarean section. The cloned Labrador retrievers were genetically identical to the nuclear donor dog. The body weight of clone-1, -2, -3, and -4 was increased from 0.66, 0.40, 0.39, and 0.37 kg at birth to 6.2, 6.6, 6.2, and 6.0 kg at 8 weeks of age, respectively. Although clone-4 had lower numbers of RBC than reference range, the most of RBC and WBC related heamatologic results of cloned puppies were not different when compared to reference range. In serological analysis, Glucose, ALP and inorganic phosphate level of four cloned puppies was significantly higher than the reference ranges. However, there was no significant difference among four cloned dogs. This study suggests that cloned puppies derived from SCNT did not have remarkable health problems, at least in the growth pattern and hematological and serological parameters.

• Journal of Veterinary Clinics
• /
• v.27 no.4
• /
• pp.336-342
• /
• 2010

The purpose of this study was to determine the antagonistic effects of flumazenil on anesthesia induced with tiletamine/zolazepam in dogs. The anesthetic effects (sedation, analgesic, muscle relaxation, posture and auditory response score), vital signs (heart rate, respiratory rate and rectal temperature) and blood biochemistry (glucose (GLU), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST)) were examined as indicators of the antagonistic effects. A total of 6 clinically healthy mongrel dogs were used in this study. The dogs in TZ group received administration of tiletamine/zolazepam 10 mg/kg IV. The dogs in TZF group received administration dose of TZ 10 mg/ kg IV followed by the administration of flumazenil 0.1 mg/kg 20 minutes after administering a TZ 10 mg/kg dose. There were significant differences in the recovery of anesthesia between the groups. The GLU level in the TZF group after the administration of flumazenil was significantly higher than that of the TZ group. There was a larger change in the HR in the TZF group than in the TZ group until 30 minutes after flumazenil administration. The sternal recumbency, standing and walking times of the TZF group were faster than those of the TZ group. In conclusion, flumazenil showed antagonistic effect against tiletamine/zolazepam in dogs. When recovering from anesthesia, flumazenil reduced sternal recumbency, standing and walking times.

• Journal of Veterinary Clinics
• /
• v.32 no.4
• /
• pp.352-355
• /
• 2015

This study was conducted to ascertain whether the scent detection ability of a donor dog having extraordinary talent in cancer detection can be conserved through cloning. A specially trained dog for colorectal cancer detection was cloned, and she was trained and tested to detect breast cancers using breath samples collected from patients and healthy volunteers. Scent detection sensitivity of the clone was 93.3% and specificity was 99.5%, similar with those of donor (91% and 99%). Furthermore, the clone successfully detected early stage of breast cancers. Therefore, superior canine scent detection ability for cancer screening could be preserved through cloning.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2001.11a
• /
• pp.102-102
• /
• 2001

Taurine (2-ethaneaminosulfonic acid) is one of the major intracellular ${\beta}$ -amino acids in mammals and is required for a number of biological processes including membrane stabilization, osmoregulation, antioxidation, detoxification, modulation of calcium flux and neurornodulation. The taurine transporter (TAUT) which contains 12 hydrophobic membrane-spanning domains has been cloned from dog kidney, rat brain, mouse brain, human thyroid, placenta and retina. In this study, The TAUT cDNA from the human intestinal epithelial cell, HT-29 was cloned and sequenced. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify partial cDNA encoding human intestinal TAUT. The coding region of the PCR product was 732 bp long. The primers were designed to encode highly conserved amino acid sequences near the transmembrane domains III (IPYFIFLF) and Ⅵ (KYKYNSYR) both in human and mouse. The TAUT cDNA amplified was ligated into the pGEX 4T-1 expression vector. The resulting sequence of human intestinal TAUT cDNA (Accession number of NCBI Genebank is AF346763) was identical to the sequences of the TAUTs previously determined in the human placenta and retina except 3 base pairs from that of the reported human thyroid. TAUT specific antibodies were generated to use them as biological tools in the studies of the biological role of TAUT. Peptides of 149-162 amino acid residue (14 amino acids) of the TAUT were synthesized. The synthetic peptide used in this study was LFQSFQKELPWAHC. This region was chosen not only to avoid putative glycosylation sites but also to exclude regions of known homology with GABA transporters in the extracellular hydrophilic domains. The synthetic peptide, TAUT-1 was conjugated with carrier protein, kehole lympet hemocyanin (KLH) to use as an antigen. When used for immunization on a rabbit to produce polyclonal antiserum, the conjugates elicited high -titered specific anti-TAUT-1 antibodies, which reacted well with the ovalbumin (OVA) conjugated peptides in ELISA. The KLH-conjugated peptide was also used as immunizing antigen in BALB/c mice to produce TAUT specific monoclonal antibodies. From the culture supernatant of the hybridoma, the specificity of anti-TAUT-1 monoclonal antibodies was confirmed by ELISA. Further applications of more tools in TAUT expression analysis will be performed such as western blotting and flow cytometry.

• Journal of Veterinary Clinics
• /
• v.22 no.3
• /
• pp.264-267
• /
• 2005

A 12-year-old, 8.0 kg, spayed female, mixed-breed dog was presented to the Veterinary Medical Teaching Hospital of Chungnam National University (VMTH, CNU). That case has been growing up mass in her left upper hindlimb about for 2 years and has showed vomiting and anorexia for 3 days. The patient was diagnosed with mast cell tumor on the basis of fine-needle aspiration (FNA) cytology techniques. According to World Health Organization clinical staging system for diagnosing mast cell tumors, it was classified into stage IIIa. The patient was treated by adjuvant corticosteroid therapy, but complete surgical excision was not achieved by owner's request. In the early stage of therapy, the size of the mass was gradually reduced with only adjuvant glucocorticoid therapy, so the patient's general condition was maintained well. But after 53 days later, the treatmant was not effective anymore and mass size was increased. Two months later, she was euthanized because of intermittent vomiting and severe respiratory distress. Splenic mass, duodenal ulceration, liver mass and infiltrated mast cell tumor in upper hindlimb muscle region were found in necropsy examination.