PG102 may have potential as a safe and effective reagent for the prevention or treatment of asthma.

cAd-MSC have higher stem-cell potential than cFF in terms of proliferation patterns, epigenetic modification and pluripotency, thus cAd-MSC could be more appropriate than cFF as donors of nuclei in somatic cell nuclear transfer for transgenesis.

The pathogenesis of neuronal degenerative disease as Alzheimer's disease (AD) has been a subject of intensive research for the last few decades worldwide. But despite such effort, treatment or preventive measures for AD have so far made no breakthrough. One of the contributing factors that hindered the progress of research is the lack of appropriate AD models. Mouse models have limitations for AD research because the irreconcilable species gap between the rodent and human has impeded the research itself as well as the application of the findings from the rodent studies to human cases. As an alternative, here we performed a preliminary study to develop novel neuronal degenerative disease models using a canine transgenic somatic cell nuclear transfer (SCNT) technology. The aim of this study is to produce a transgenic dog that expresses neuron-specific transgene in the brain by SCNT. In this study, we chose human synapsin 1 promoter as primarily neuron selective, driving the red fluorescent protein transgene. For SCNT, synapsin 1-red fluorescence protein (SYN1-RFP) was introduced into female beagle adipose-derived stem cell via lentiviral vector infection. The SYN1-RFP cells were injected into enucleated in vivo-matured dog oocytes and fused by electric stimulation. The fused couplets (80/94, 85.1%) were chemically activated and transferred into the uterine tube of 5 naturally oestrus-synchronized surrogates. Three of them (60%) maintained pregnancy and subsequently gave birth to 3 cloned pups (SYN1-RFP A, SYN1-RFP B, SYN1-RFP C) by natural delivery or cesarean section. Birth weights of the offspring ranged from 120 to 280 g and SYN1-RFP C is still alive, healthy and does not show any abnormalities. The microsatellite analysis shows that all SYN1-RFP puppies originated from the SYN1-RFP cells used in SCNT and mitochondrial DNA analysis shows that the puppies had been derived from the oocyte donors. In order to investigate the result in multiple transgene insertions, SYN-RFP puppies were screened by Southern blot analysis using DNA extracted from skin biopsies. Transgene copy number was estimated by Southern blot analysis. The SYN-RFP A and B that died at 3 days after birth had approximately 5 and 2 copies of the transgene integrated, respectively, whereas the alive SYN-RFP C has 1 copy. SYN-RFP B was particular in that it did not express RFP in the entire body, but samples collected postmortem showed expression of the RFP transgene under the human synapsin 1 promoter in neural cells in the brain of SYN-RFP B. In conclusion, we report here that (1) the human synapsin promoter is functional in neural cells of dog brain and (2) a neural-specific-transgene-expressed dog was generated for the first time by transgenic SCNT technique. Furthermore, the SYN-RFP dog has great potential to understand the function of a neuronal degenerative disease model dog. This study was supported by MKE (Grant # 10033839-2011-13), RNL Bio, IPET and TS Corporation.

Canine somatic cell nuclear transfer (cSCNT) has been used as a useful tool for propagation of elite working dogs. In 2009, 7 cloned dogs were successfully produced using somatic cells derived from the excellent drug-sniffing dog of Korea Customs Service. All cloned dogs perfectly performed drug detection in Incheon International Airport. The objective of the present study was to compare the efficiency of the 2 activation culture media to clone the retired Baekdu, a veteran rescue dog that performed lifesaving activities worldwide for 6 years in Korea National Emergency Management Agency (NEMA). Ear tissue was collected from a 10-year-old male German Shepherd and fibroblasts were cultured for cSCNT. The cells were injected into the perivitelline space of enucleated in vivo-matured dog oocytes, fused with electric stimulation using an electro cell fusion apparatus (Nepa Gene Co. Ltd.), and activated chemically. In the activation protocol, 2 different types of media were tested to investigate the effect of proteins with undefined functions. The first medium was a modified synthetic oviduct fluid (mSOF), which is a complex culture medium with BSA that includes undefined functions. The second medium was the porcine zygote medium (PZM-5), which is a chemically defined medium with polyvinyl alcohol (PVA). The fused couplets were activated by mSOF medium supplemented with 1.9 nM DMAP (SOF-DMAP), and PZM-5 supplemented with 1.9 nM DMAP (PZM-DMAP) for 4 h, followed by 4 min of calcium ionophore treatment. Then, reconstructed oocytes were transferred into the uterine tube of naturally estrus-synchronized surrogate dogs. In the PZM-DMAP group, a total of 56 activated cloned embryos were transferred into 3 female recipient dogs, and a total of 64 activated cloned embryos from the SOF-DMAP group were transferred into 4 female recipients. Pregnancy diagnosis was performed using a SONOACE 9900 (Medison, Seoul, Korea) ultrasound scanner with 7.0-MHz linear-array probe between 30 and 35 days after embryo transfer. As a result, pregnancy was detected in 1 out of 3 surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and 1 pregnancy (25%) was detected in 4 surrogate mothers receiving cloned embryos from the SOF-DMAP group. Two pregnant dogs each gave birth to 1 healthy cloned puppy by cesarean section. This study shows that existence of proteins with undefined functions in activation medium did not affect the dog cloning. In addition, the number of elite working dogs in diverse fields can be increased by the NT technique using donor cells derived from small tissue of elite working dogs. This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, and TS Corporation.

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.