The promise of dog cloning

Dog cloning as a concept is no longer infeasible. Starting with Snuppy, the first cloned dog in the world, somatic cell nuclear transfer (SCNT) has been continuously developed and used for diverse purposes. In this article we summarise the current method for SCNT, the normality of cloned dogs and the application of dog cloning not only for personal reasons, but also for public purposes.

37 NORMALITY OF NEONATAL REFLEX IN CLONED DOGS

Since the birth of the world’s first cloned dog, Snuppy, somatic cell nuclear transfer (SCNT) has been a useful tool to propagate the dogs with identical genetic information. However, it is known that cloned animals sometimes exhibit phenotypic instability or abnormality. There have been few investigations about the normality of the neonatal reflex in cloned animals. Therefore, the objective of this study was to evaluate the neonatal reflex in 3 breeds of cloned dogs including shepherd, retriever, and beagle from birth to 28 days of age. Through SCNT, 8 cloned dogs were produced. After birth, 3 types of neonatal reflexes were examined and scored. For examining the flexor dominance reflex, neonatal cloned dogs were held upright and the flexor position of the limb was scored. To evaluate the withdrawal and crossed extensor reflexes, neonates were placed in lateral recumbence and their forelimbs were allowed to relax. Then, the distal forelimbs were pinched and responses were scored according to the frequency and intensity (strong = score 2, variable = score 1, and absent = score 0). The standard responses of neonates were referred from Lindsay et al. (2000 Handbook of Applied Dog Behavior and Training 1, 31–47). Descriptive analysis was used, which was based on the scores from 3 referees who evaluated the videos. The flexor dominance reflex could not be observed (score 0.0) in shepherd by Day 8, in beagle by Day 14 and in retriever by Day 16. Withdrawal reflex began to decrease on Day 22 with score 1.8 for beagle and retriever but decreased in shepherd starting on Day 24 with score 1.8. Crossed extensor reflex for shepherd started to disappear on Day 14 with score 1.5 and completely disappeared (score 0.0) on Day 22; for beagle started to disappear on Day 16 with score 1.8 and was still found until Day 28 with score 1.1; for retriever started to disappear on Day 20 and 28 with score 1.7 and 0.7, respectively. Flexor dominance reflex disappeared in cloned shepherd at a similar time to standard but beagle and retriever seem delayed 6 to 8 days compared with the reference. Withdrawal reflex in all breeds showed normal changes that should persist until adulthood. Cross extensor reflex in shepherd was close to reference but in beagle and retriever was delayed beyond Day 28; this reflex should disappear before adulthood. This study demonstrated that normal neonatal reflexes were identified in the cloned dogs, with some variations among breed. To adapt neonatal reflex as a marker to confirm phenotypic normality in cloned dogs, further investigation using various breeds of cloned dogs and greater numbers of subjects is needed. This study was supported by IPET (#316002-05-1-SB010), RDA (#PJ010928032016), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.

36 EFFECT OF SUBEROYLANILIDE HYDROXAMIC ACID TREATED DONOR CELLS ON DOG CLONING

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACi), on dog cloning efficiency. Relative mRNA expression of the bax1, bcl2, and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 μM) of SAHA and durations (0, 20, 44 h) were assessed using real-time polymerase chain reaction. After determining an optimum concentration and duration, histone acetylation levels (H3K9, H4K5/K8/K12/K16) of SAHA-treated cells were analysed using immunostaining. The SAHA-treated cells were used as donor cells for somatic cell nuclear transfer, and activated reconstructed embryos were transferred to recipients. Pregnancy diagnosis was performed by ultrasonography at least 29 days after the embryo transfer. All experiments were repeated more than 3 times and the data were analysed using Graph Prism software (GraphPad Software Inc., San Diego, CA, USA). An unpaired t-test was used to compare transcripts levels and fluorescence intensities. A chi-squared test was used to compare the implantation rates. The bax1/bcl2 ratio of the 1 μM SAHA group was similar to that of control but significantly increased in the 10 μM and 50 μM groups. Expression of Dnmt1 was decreased in the 1 μM SAHA group, and the 10 μM and 50 μM groups showed the lowest expression compared with the control group. Although the bax1/blc2 ratio was not affected by the SAHA treatment duration, 20-h treatment group showed significantly decreased Dnmt1 levels compared with control group. As a pan-HDAC inhibitor, 1 μM for 20 h of SAHA treatment significantly increased acetylation of H3K9, H4K5, H4K8, and H4K16. For control and SAHA groups, a total of 76 and 64 cloned embryos were produced and transferred to 7 and 5 recipients, respectively. Three fetuses were diagnosed in both groups but there was no significant difference in the pregnancy rate. In conclusion, although SAHA treatment as used in this study significantly decreased bax/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 and H4 acetylation, it would not enough to increase in vivo developmental competence of cloned dog embryos.This study was supported by RDA (#PJ008975022014), IPET (#311062–04–3SB010), Research Institute for Veterinary Science and the BK21 plus program.

37 RESTORING REPROGRAMMING ABNORMALITIES IN A CLONED DOG HAVING ECTOPIC LIVER AND GALL BLADDER BY RECLONING

The risk of reprogramming abnormalities such as placental hyperdevelopment, excessive fetal growth, or abnormalities of the immune system in cloned neonates is one of the major concerns in cloning research. However, until now, relatively few studies about birth defects have been reported in dog cloning, which might be due to the short in vitro manipulated procedure in this species. Here, we report a cloned dog having abnormal liver and investigated whether the abnormal liver was due to genetic modification. A cloned beagle was produced from a fibroblast derived from a 10-year-old donor, but accidentally died due to cannibalism of a nanny dog on the day of birth. During autopsy, an abnormal liver structure was found; 7 lobes were presented at the normal liver position inside the abdomen, but there was no gall bladder. Interestingly, 3 additional lobes with a gall bladder were found in between the rib and the skin. There were no other macroscopic anomalies observed in this puppy. To evaluate the heredity of this liver abnormality, the liver structure of the donor dog was diagnosed by computed tomography (CT). Also, to assess the possibility of restoring the liver abnormality, recloning was performed using a fibroblast cell line established from the dead pup, and liver positions in the recloned dogs were diagnosed by CT after puberty. In results, 2 recipients delivered 5 recloned dogs with birthweights of 510, 250, 460, 400, and 410 g. The smallest one showed severe bow-legged phenotype in its hind leg, and a unique coat pattern that showed the largest white coat surface. The pup died 10 days after birth, and no other abnormal phenotype was found during autopsy. The other 4 pups showed normal morphology at birth. The CT results showed normal positioning of the liver and gall bladder in all experimental dogs, including the original cell donor dog and recloned dogs. To our knowledge, there have been no reported cases of an ectopic liver and gall bladder present between rib and skin in both cloned and noncloned animals, and we consider that these abnormalities are not a due to genetic cause. Further studies regarding aberrant epigenetic reprogramming in the abnormal liver formation are needed.