Joung Joo Kim

Birth of Beagle dogs by somatic cell nuclear transfer.

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.

Cloning Missy: Obtaining Multiple Offspring of a Specific Canine Genotype by Somatic Cell Nuclear Transfer

The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy‐six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase‐like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family. Mol. Reprod. Dev. 76: 483–489, 2009.

Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase

Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.

Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines

Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P < 0.01). Perinatal mortality until weaning was significantly higher in small breeds (33.3%) compared with medium, large, or ultra-large breeds where no mortality was observed. The mean birth weight of cloned pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype.

Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XYDSD gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XYDSD in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein

Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimers disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and β-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.

Relationship between time post-ovulation and progesterone on oocyte maturation and pregnancy in canine cloning

Canine oocytes ovulated at prophase complete meiosis and continue to develop in presence of a high progesterone concentration in the oviduct. Considering that meiotic competence of canine oocyte is accomplished in the oviductal environment, we postulate that hormonal milieu resulting from the circulating progesterone concentration may affect oocyte maturation and early development of embryos. From 237 oocyte donors, 2620 oocytes were collected and their meiotic status and morphology were determined. To determine optimal characteristics of the mature oocytes subjected to nuclear transfer, a proportion of the meiotic status of the oocytes were classified in reference to time post-ovulation as well as progesterone (P4) level. A high proportion of matured oocytes were collected from >126h (55.5%) post-ovulation or 40-50ngmL-1 (46.4%) group compared to the other groups. Of the oocyte donors that provided mature oocytes in vivo, there was no correlation between serum progesterone of donors and time post ovulation, however, time post-ovulation were significantly shorter for <30ng/mL group (P<0.05). Using mature oocytes, 1161 cloned embryos were reconstructed and transferred into 77 surrogates. In order to determine the relationship between pregnancy performance and serum progesterone level, embryos were transferred into surrogates showing various P4 serum levels. The highest pregnancy (31.8%) and live birth cloning efficacy (2.2%) rates were observed when the embryos were transferred into surrogates with circulating P4 levels were from 40 to 50ngmL-1. In conclusion, measurement of circulating progesterone of female dog could be a suitable an indicator of the optimal time to collect quality oocyte and to select surrogates for cloning.