Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.     

小鼠体内卵母细胞死亡的形态变化研究

目前大多数研究主要是针对体外的卵母细胞凋亡,并且通常采用抗癌试剂来人为的引入。然而,对体内卵母细胞死亡的过程还不是很明确。为了查明体内未受精卵的死亡过程,我们检测了从输卵管壶腹部冲出的注射了HCG后不同间隔的卵母细胞中的细胞化学物质的改变情况。在每一个收集的时间段,在显微镜下将收集的卵母细胞从表观上分成四组:单细胞卵母细胞(没有激活和没有核或胞浆移动),激活的卵母细胞(带有核的单细胞,2-或4-细胞),碎裂的卵母细胞及其死亡的卵母细胞。随着时间的推移,单细胞卵母细胞的数量下降,死亡的卵母细胞数量增加,但是激活或碎裂的卵母细胞数量未发生改变。同时,检测到的大多数死亡的卵母细胞都是单细胞。在每一个时间点上,用抗微管蛋白抗体对单细胞卵母细胞进行着色,来检测它们的纺锤体状态。在注射HCG24h之后,大多数超排后的卵母细胞都有两极纺锤体,然而在超排后64h的单细胞卵母细胞就没有纺锤体。结果表明,大多数的体内卵母细胞在单细胞期就出现死亡,而激活或碎裂的卵母细胞则不是。     

Surveillance of classical swine fever in wild boar in South Korea from 2010–2014

Classical swine fever (CSF) is a highly contagious systemic hemorrhagic viral disease of pigs. Wild boar plays a crucial role in the epidemiology of CSF. Between 2010 and 2014, samples were collected nationwide from 6,654 wild boars hunted in South Korea. Anti-CSF antibodies were identified in 0.59% (39 of 6,654) of the wild boar samples using a virus neutralization test and were primarily detected in wild boars living close to the demilitarized zone and the area of the Taebaek Mountains surroundings. The CSF virus (subgroup 2.1b) was isolated from two wild boars captured in a nearby border area. The criteria used to define high-risk areas for targeted CSF surveillance in South Korea should be further expanded to include other regions nationwide.     

Cardiorespiratory and anesthetic effects produced by the combination of butorphanol,medetomidine and alfaxalone administered intramuscularly in Beagle dogs

This study evaluated anesthesia quality, degree of analgesia and cardiorespiratory parameters after intramuscular (IM) injection of a combination of butorphanol (0.1 mg/kg), medetomidine (10 µg/kg) and alfaxalone (1.5 mg/kg) in ten healthy adult Beagle dogs. Rectal temperature (T), heart rate (HR), respiratory rate (f_R), arterial pressure, arterial blood gases and M-mode echocardiographic left ventricular (LV) indices were measured before drug administration and every 10 min thereafter until extubation. Mean duration of anesthesia, recovery and analgesia were 89 ± 17, 6 ± 1 and 80 ± 12 min. HR, f_R, partial pressure of arterial CO₂ and O₂, arterial pressure, and LV contractility were significantly altered during anesthesia. IM administration of the drug combination provided acceptable anesthesia, but produced substantial cardiorespiratory suppression.     

Diagnosis of a large splenic tumor in a dog: computed tomography versus magnetic resonance imaging

This study demonstrated magnetic resonance imaging (MRI) and computed tomography for large-sized splenic hemangiosarcoma. Radiography and ultrasonography revealed the presence of a large-sized soft-tissue mass in the cranial abdomen. Computed tomography showed hypoattenuating mass. The mass was located in contact with liver, spleen and stomach, and the origin of the mass remained ambiguous. The mass was T2-hyperintense and T1-hypointense with mild contrast enhancement. MRI allowed a differentiation between large-sized tumor and neighboring normal structure, and the mass was consequently identified as arising from spleen. These results suggested that MRI might be a useful tool to visualize large-sized splenic tumors and improve the accuracy of diagnosis.     

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.     

Collagen complexes increase the efficiency of iPS cells generated using fibroblasts from adult mice

Different interventions are being tested for restoration of the youthfulness of adult mouse-derived fibroblasts. However, fundamental issues, such as the decline of adult mouse-derived fibroblast activity with age, remain unresolved. Therefore, in this study, we examined whether treatment with collagen complexes has beneficial effects on the rejuvenation or reprogramming of adult mouse-derived fibroblasts. Further, we investigated the mechanisms of rejuvenation of adult mouse-derived fibroblasts during treatment with total collagen complexes. We isolated total collagen complexes from the tails of young mice and cultured adult mouse-derived fibroblasts with or without the collagen complexes. When compared with fibroblasts cultured without collagen complexes, adult-derived fibroblasts cultured with collagen complexes over five consecutive passages showed a more youthful state, expanded at a higher rate, and exhibited reduced spontaneous cell death. The fibroblasts cultured in the presence of collagen complexes also showed extensive demethylation in the promoter regions of cell cycle-related genes such as PCNA, increased proliferation, and decreased senescence. In addition, the efficiency of reprogramming of fibroblasts to become induced pluripotent stem (iPS) cells was significantly higher in young- and adult-derived fibroblasts cultured with collagen complexes than in adult-derived fibroblasts cultured alone. Furthermore, mechanistic evidence shows that genes involved in anti-proliferative pathways, including Ink4a/Arf locus genes and p53, were downregulated in fibroblasts exposed to collagen complexes. Interestingly, our results suggest that the rejuvenation process was mediated via the α2β1 integrin-dependent Bmi-1 pathway. Thus, collagen complexes both stimulate proliferation and inhibit cell death and growth arrest in fibroblasts, which appears to be a promising approach for improving the efficiency of reprogramming.     

Nonsurgical resolution of caudal mediastinal paraesophageal abscess in a cat

A one-year-old, castrated male domestic short hair cat was admitted with a history of anorexia, regurgitation and pyrexia for two days. Fever and leukocytosis were identified. There were a large soft tissue density oval mass in the caudal mediastinum on thoracic radiographs, a fluid-filled oval mass in the caudal mediastinum on ultrasonography, and left-sided and ventrally displaced and compressed esophagus on esophagram. On esophageal endoscopy, there were no esophageal abnormalities. CT findings with a fluid filled mass with rim enhancement indicated a caudal mediastinal paraesophageal abscess. The patient was treated with oral antibiotics, because the owner declined percutaneous drainage and surgery. The patient was admitted on emergency with severe respiratory distress; and ruptured abscess and deteriorated pleuropneumonia were suspected. With intensive hospitalization care and additional antibiotic therapy, the patient had full recovery.     

Characterization of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from pigs presenting with diarrhea in Korea

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans.