Cleft palates with or without cleft lip is one of the most common congenital craniofacial defects in dogs. It has been reported that monogenic autosomal recessive inheritance caused this defect in this species. However, here, we aimed to report cleft palate in a cloned dog. A fibroblast cell line was established from skin tissues of an eight-year-old German shepherd dog. Blood was collected from oocyte donor dogs, and serum progesterone concentration was measured by chemiluminescence enzyme immunoassay method. Ovulation was determined when serum progesterone results reached 5-10 ng/ml, and in vivo matured oocytes were collected surgically about 72 hr after ovulation. Donor cells were cultured with Dulbecco’s modified Eagle medium supplemented with 10% (v/v) fetal bovine serum until confluence. An in vivo matured oocyte was enucleated, and a donor cell was injected into the perivitelline space. The oocyte-cell couplet was electrically fused, and chemically activated. Reconstructed embryos were transferred to an oviduct of a recipient. Pregnancy diagnosis was performed 27 days after the embryo transfer, and ultrasonography of fetal heart beat, and rectal temperature and serum progesterone value of recipient was monitored until the day of delivery. Microsatellite analysis was performed using genomic DNA of cell donor, clones, and oocyte donors. As results, a total of 74 cloned embryos were transferred to five recipients, and one recipient diagnosed as pregnant with two fetuses by ultrasonography and radiology. Caesarean section was performed on day 58 after embryo transfer due to a decreased heart beat of a fetus, which was lower than 180. Two cloned puppies with 640g and 320g of birth weight were delivered safety, but the small one was born with a cleft palate. Microsatellite analysis results of both clones were identical with the cell donor. Cleft palate of the clone was surgically corrected on day 40 after birth. To our knowledge, there has been no report about cleft palate in cloned dogs, and also, no report about clones with different phenotype of cleft palate in dogs. Therefore, this study can give a clue of cleft palate in dogs, which might not be a genetic cause. Further studies about aberrant epigenetic reprogramming in those clones are needed.

The canine major histocompatibility complex (MHC) is referred to dog leukocyte antigens (DLA), which is known to be the most polymorphic genetic system in canine species. Many cloned dogs have been produced since Snuppy, first cloned dog, there was no research about genetic identity of MHC among cloned animals. Recently in Lee’s group, two non-transgenic cloned beagles (BG1, 2) were produced by somatic cell nuclear transfer (SCNT) using fetal fibroblast (BF). Also, four transgenic cloned beagles (Ruppy 1-3, 5) were generated using transgenic BF transfected with Red fluorescent protein (RFP) gene. We hypothesize that non-transgenic (BG1, 2) and transgenic (Ruppy 1-3, 5) cloned beagles derived from identical donor cells have the same immunological genetic characteristic except for RFP gene insertion in the genome. Thus, the aim of this study is to confirm the immunological identity of DLA class II in cloned beagles produced using same nuclear donor cell. Genomic DNA was extracted from blood of BG1, BG2, Ruppy 1, 2, 3 and 5. Genomic DNA of normal two control beagle, no correlation with BF was also investigated for rulling out the possibility that beagles were inbred. Forward and reverse primers used for DLA-DQA1 and DQB1 respectively were DQAF: 5’-TAAGGTTCTTTTCTCCCTCT-3’ and DQAR: 5’-GGACAGATTCAGTGAAGAGA-3’ DQBR:5’-CTCACTGGCCCGGCTGTCTC-3’ and DQBR: 5’-CACCTCGC CGCTGCAACGTG-3’. Polymerase Chain Reaction (PCR) products were purified, sequenced directly using the Big Dye Terminator kit. Sequencing analysis was performed on an automated 3730xl DNA analyzer. In experiment 1, sequence of DLA-DQ alpha 1 (DQA1) and DLA-DQ beta 1 (DQB1) exon 2, hypervariabel region, was compared in BG1 and BG2. Experiment 2 also compared the sequence of DQA1 and DQB1 among Ruppy 1, 2, 3 and 5. Experimental 3 compared sequence of DQA1 and DQB1 among all cloned dogs (BG1, BG2 and Ruppy 1-3, 5). As a result, BG1 and BG2 have same allele for DQA1 and DQB1 as we expected. They share DQA1*00101 and DQB1*02901 in experiment 1. In experiment 2, Ruppy 1, 2, 3 and 5 also have identical DQA1*00101 and DQB1*02901 allele. No discrimination between transgenic dogs and cloned dogs was seen in DQA1 and DQB1 Allele in experiment 3. DQA1, DQB1 allele was identified as *00101 and *02901 in all dogs. We provided the allele identity of DQA1and DQB1 in cloned beagles, which can be used as preliminary data for immunological related studies. In conclusion, transgenic cloned dogs despite of red fluorescent protein genes being inserted in their nuclear DNA were immunologically compatible with non-transgenic cloned dogs. We demonstrated that cloned beagles produced using identical nuclear donor were immunologically compatible.

Controllable transgenic expression systems in transgenic animal model are valuable to the development of therapeutic approaches in human medical fields. The aim of this study was to 1) produce a transgenic cloned dog using inducible tetracycline vector system, and 2) investigate whether the transgenic cloned dog could be induced the transgene expression using doxycycline (Doxy). Canine fetal fibroblasts were infected with retroviral vectors designed to express the enhanced green fluorescent protein (eGFP) gene under the control of tetracycline-inducible promoter. For somatic cell nuclear transfer (SCNT), nucleus of an in vivo matured oocyte was removed and an eGFP expressed cell cultured with 1 ㎍/㎖ of Doxy was injected. After electrical fusion and chemical activation, the reconstructed embryos were transferred to a recipient and pregnancy diagnosis was performed by ultrasonography. Experiment I evaluated the mean fluorescence intensity (MFI) of infected cells while the cells were cultured in the presence of 1 ㎍/㎖ of Doxy for 5 days, and then in the absence of Doxy for 7 days using fluorescence-activated cell sorter. Experiment II was designed to produce an eGFP controllable transgenic cloned dog via SCNT. For verification of transgenic dog, experiment III was performed Southern Blot analysis and observation in vivo regulation of eGFP expression in the cloned dog treated with 100 ㎎/㎏ of Doxy every 2 days for 2 weeks under ultraviolet light. In experiment IV, western blot was used to detect eGFP increase and decrease in skin tissues of transgenic dog under the presence or absence of Doxy. In the results of experiment I, the MFI for infected cells was rapidly increased to approximately 42.3 times after 3 day-treatment compared to pre-treatment and quickly decreased 3 days after ceasing the treatment. In experiment II, a total of 203 embryos were transferred to nine recipients and three pregnant delivered three pups (Tet-on eGFP 0, Tet-on eGFP 1, and Tet-on eGFP 2) by C-sec and Tet-on eGFP 2 among them is still alive. All cloned pups were genetically identical to the donor cell. Tet-on eGFP 2 showed an apparent in vivo eGFP expression on her body after Doxy administration in experiment III. The result of Sothern blotting showed that the transgene insertion was detected from the three cloned dogs and all organs of Tet-on eGFP 1. Experiment IV indicated that a robust eGFP expression in skin tissue of Tet-on eGFP 2 rapidly increased after Doxy treatment and gradually decreased to basal level on 9 weeks after ceasing the treatment. In conclusion, we report here for the first time an inducible transgenic system in canine species and it can stably induce the transgene expression at intended time. This study has demonstrated the capacity to generate transgenic model dog which could regulate the transgene and it would contribute to human medical research fields.