Genetic and morphometric characteristics of Korean wild mice (KWM/Hym) captured at Chuncheon, South Korea / 한국실험동물학회지

Laboratory inbred mice are used widely and commonly in biomedical research, but inbred mice do not have a big enough gene pool for the research. In this study, genetic and morphometric analyses were performed to obtain data on the characteristics of a newly developing inbred strain (KWM/Hym) captured from Chuncheon, Korea. All of five Korean wild male mice have the zinc-finger Y (ZfY) gene. Also, all of 19 Korean wild mice used in this analysis have the AKV-type murine leukemia virus gene, indicating that Korean wild mice might be Mus musculus musculus. To identify the genetic polymorphism in KWM/Hym, SNP analysis was performed. In a comparison with 28 SNP markers, there was a considerable difference between KWM/Hym and several inbred strains. The homogeneity between KWM/Hym and the inbred strains was as follows: C57BL/6J (39.3%), BALB/c AJic (42.9%), and DBA/2J (50%). KWM/Hym is most similar to the PWK/PhJ inbred strain (96.4%) derived from wild mice (Czech Republic). To identify the morphometric characteristics of KWM/Hym, the external morphology was measured. The tail ratio of male and female was 79.60±3.09 and 73.55±6.14%, respectively. KWM/Hym has short and agouti-colored hairs and its belly is white with golden hair. Taking these results together, KWM/Hym, a newly developing inbred mouse originated from wild mouse, might be use as new genetic resources to overcome the limitations of the current laboratory mice.

Over-expression of myosin7A in cochlear hair cells of circling mice / 한국실험동물학회지

Circling mouse (C57BL/6J-cir/cir) deleted the transmembrane inner ear (Tmie) gene is an animal model for human non-syndromic recessive deafness, DFNB6. In circling mouse, hair cells in the cochlea have degenerated and hair bundles have become irregularity as time goes on. Tmie protein carries out a function of the mechanoelectrical transduction channel in cochlear hair cells. Myosin7a (MYO7A) protein has key roles in development of the cochlear hair bundles as well as in the function of cochlear hair cells. To find whether Tmie protein interacts with MYO7A proteins in the cochlea postnatal developmental stage, we investigated expression of the MYO7A proteins in the cochlear hair cells of circling mice by western blot analysis and whole mount immunofluorescence at postnatal day 5 (P5). The expression of MYO7A showed statistically significant increase in the cochlea of C57BL/6J-+/cir and C57BL/6J-cir/cir mice than that of C57BL/6J-+/+ mice. The MYO7A intensity of the cochlear hair cells also increased in C57BL/6J-+/cir and C57BL/6J-cir/cir mice compared with those of C57BL/6J-+/+ mice. Taken together, the results indicate that Tmie protein may have an important role with MYO7A protein in the development and maintenance of the stereociliary bundles during postnatal developmental stage of the cochlea.

Rapid and efficient identification of the mouse leptin receptor mutation (C57BL/KsJ-db/db) by tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) analysis / 한국실험동물학회지

The C57BLKS/J-Lepr(db) mouse has a point mutation in the leptin receptor gene and is one of the most useful animal model for non-insulin dependent diabetes mellitus in human. Since the homozygote of C57BLKS/J-Lepr(db) mouse is infertile, detection of point mutation in the leptin receptor gene is important for efficient maintaining strains as well as mass production of homozygotes. To develop a rapid and efficient genotyping method for C57BLKS/J-Lepr(db) mouse, the tetra-primer amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) was used. The 407 and 199 bp PCR products were amplified from normal (+/+) mice; while the 407 and 268 bp PCR products were amplified from homozygotes (db/db) mice; and the 407, 268, and 199 bp PCR products were amplified from heterozygotes (db/+) mice. This result showed that the tetra-primer ARMS-PCR analysis by us is suitable to detect point mutation of the leptin receptor gene. Taken together, our method will dramatically reduce animal use for maintenance of strains as well as production of homozygote in the C57BLKS/J-Lepr(db) strains.

Erratum: Rapid and efficient identification of the mouse leptin receptor mutation (C57BLKS/J-Lepr(db)) by tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) analysis / 한국실험동물학회지

The published article includes an error in Figure 1.

Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.

Surgical Outcome of Urethroplasty Using Penile Circular Fasciocutaneous Flap for Anterior Urethral Stricture

PURPOSE: Penile circular fasciocutaneous flap urethroplasty is a useful technique for a long anterior urethral stricture due to the flap's hairless nature and ample length. We investigated the surgical outcomes of urethroplasty for a complex anterior urethral stricture, performed using a penile circular fasciocutaneous flap. MATERIALS AND METHODS: Between 2008 and 2013, we performed a retrospective review of 29 patients who underwent urethroplasty using a penile circular fasciocutaneous flap and had at least 6 months of follow-up. A total of 20 cases utilized only a fasciocutaneous flap, while 9 cases combined a fasciocutaneous flap with other surgery. Success was defined as no requirement of additional urethral instrumentation. RESULTS: The overall success rate was 68.9% (20 out of 29 cases) at a median follow-up of 19 months. Furthermore, fasciocutaneous flap urethroplasty rendered the actual stricture-free rate of 79.3%. The location of recurrence was mostly at the junction of the flap. Among 9 surgical failures, 5 cases were treated successfully by using an additional surgical procedure. Fistula repair was needed in 1 case 4 months later. Further, periodic urethral dilation was performed in the remaining 3 cases. The failure rate was significantly higher in patients with suprapubic cystostomy than in patients without suprapubic cystostomy. The most common complication was post-micturition dribbling. CONCLUSIONS: Penile circular fasciocutaneous flap urethroplasty is a useful method for the reconstruction of a long anterior urethral stricture. A sufficient healthy margin should be acquired for better surgical results due to the fact that most recurrence occurs at the junction of the flap.

Surgical Outcome of Urethroplasty Using Penile Circular Fasciocutaneous Flap for Anterior Urethral Stricture

PURPOSE: Penile circular fasciocutaneous flap urethroplasty is a useful technique for a long anterior urethral stricture due to the flap's hairless nature and ample length. We investigated the surgical outcomes of urethroplasty for a complex anterior urethral stricture, performed using a penile circular fasciocutaneous flap. MATERIALS AND METHODS: Between 2008 and 2013, we performed a retrospective review of 29 patients who underwent urethroplasty using a penile circular fasciocutaneous flap and had at least 6 months of follow-up. A total of 20 cases utilized only a fasciocutaneous flap, while 9 cases combined a fasciocutaneous flap with other surgery. Success was defined as no requirement of additional urethral instrumentation. RESULTS: The overall success rate was 68.9% (20 out of 29 cases) at a median follow-up of 19 months. Furthermore, fasciocutaneous flap urethroplasty rendered the actual stricture-free rate of 79.3%. The location of recurrence was mostly at the junction of the flap. Among 9 surgical failures, 5 cases were treated successfully by using an additional surgical procedure. Fistula repair was needed in 1 case 4 months later. Further, periodic urethral dilation was performed in the remaining 3 cases. The failure rate was significantly higher in patients with suprapubic cystostomy than in patients without suprapubic cystostomy. The most common complication was post-micturition dribbling. CONCLUSIONS: Penile circular fasciocutaneous flap urethroplasty is a useful method for the reconstruction of a long anterior urethral stricture. A sufficient healthy margin should be acquired for better surgical results due to the fact that most recurrence occurs at the junction of the flap.

A high resolution genetic mapping of the faded (fe) gene to a region between D10mit156 and D10mit193 on mouse chromosome 10 / 한국실험동물학회지

The C57BL/6J-fe/fe mouse is a coat color mutant. The coat color of the homozygote mouse becomes progressively lighter with advancing age. The faded gene (fe) of C57BL/6J-fe/fe was mapped in a 2.0 cM distal to D10mit191 by our group. To make a high-resolution map, we used the Korean wild mouse (KWHM) for a backcross panel, which was captured in 1995 and has been maintained as an inbred line by our laboratory. In the inter-specific backcross panel (N=400), the fe gene was mapped to 1.0 cM distal to D10mit156. The gene order was defined: centromere -D10mit3/85 (1.3+/-0.6 cM)-D10mit155 (1.3+/-0.6 cM)-D10mit191 (2.0+/-0.7 cM)-D10mit156 (1.0+/-0.5 cM)-fe-D10mit193 (1.3+/-0.6 cM)-D10mit54 (1.0+/-0.5 cM)-D10mit44 (8.5+/-1.4 cM)-D10mit42 (10.0+/-1.5 cM). The measured distance between D10mit191 and D10mit 44 differed in both inter-specific (DBA/2) and intra-specific (KWHM) backcross panels (14.2 vs 13.8 cM). Taken together, our high-resolution linkage map of the fe locus from an intra-specific backcross panel will provide a good entry point to isolate the fe gene.

Surgical Outcome of Excision and End-to-End Anastomosis for Bulbar Urethral Stricture

PURPOSE: Although direct-vision internal urethrotomy can be performed for the management of short, bulbar urethral strictures, excision and end-to-end anastomosis remains the best procedure to guarantee a high success rate. We performed a retrospective evaluation of patients who underwent bulbar end-to-end anastomosis to assess the factors affecting surgical outcome. MATERIALS AND METHODS: We reviewed 33 patients with an average age of 55 years who underwent bulbar end-to-end anastomosis. Stricture etiology was blunt perineal trauma (54.6%), iatrogenic (24.2%), idiopathic (12.1%), and infection (9.1%). A total of 21 patients (63.6%) underwent urethrotomy, dilation, or multiple treatments before referral to our center. Clinical outcome was considered a treatment failure when any postoperative instrumentation was needed. RESULTS: Mean operation time was 151 minutes (range, 100 to 215 minutes) and mean excised stricture length was 1.5 cm (range, 0.8 to 2.3 cm). At a mean follow-up of 42.6 months (range, 8 to 96 months), 29 patients (87.9%) were symptom-free and required no further procedure. Strictures recurred in 4 patients (12.1%) within 5 months after surgery. Of four recurrences, one patient was managed successfully by urethrotomy, whereas the remaining three did not respond to urethrotomy or dilation and required additional urethroplasty. The recurrence rate was significantly higher in the patients with nontraumatic causes (iatrogenic in three, infection in one patient) than in the patients with traumatic etiology. CONCLUSIONS: Excision and end-to-end anastomosis for short, bulbar urethral stricture has an acceptable success rate of 87.9%. However, careful consideration is needed to decide on the surgical procedure if the stricture etiology is nontraumatic.

Expression of deafness protein Tmie in postnatal developmental stages of C57BL/6J mice / 한국실험동물학회지

Loss-of function mutations in the transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). Previous studies report that the circling mouse can be an animal model for DFNB6. However, the expression pattern of Tmie protein in postnatal developmental stages has not been clearly revealed. In this study we tried to investigate the expression of Tmie protein in the liver, spleen, kidney, and lung, as well as in the cochlea. We examined various tissue samples from five different age groups of C57BL/6J animals. Using western blotting analysis, the expression of Tmie protein in these organs has been identified. The results show that Tmie protein expression in the cochlea has been increased in postnatal developmental stages, indicating that Tmie plays an important role in not only the development and also in the function of the cochlea. The expression pattern of Tmie in adult mouse organs such as the liver, spleen, kidney, and spleen significantly vary in adult rats. The order of Tmie expression level in mice (63 days after birth) was spleen, liver, lung, cochlea, and kidney, whereas in the adult rat it was liver, cochlea, lung, spleen, and kidney.

Silk fibroin hydrolysate exerts an anti-diabetic effect by increasing pancreatic beta cell mass in C57BL/KsJ-db/db mice

Components of silk including silk fibroin have long been used as anti-diabetic remedies in oriental medicine. However, detailed mechanisms underlying these anti-diabetic effects remain unclear. In this study, we examined the anti-diabetic activity of silk fibroin hydrolysate (SFH) in C57BL/KsJ-db/db (db/db) mice, a well-known animal model of non-insulin dependent diabetes mellitus. When the db/db mice were administered SFH in drinking water for 6 weeks, hyperglycemia in the animals gradually disappeared and the level of glycosylated hemoglobin decreased, indicating that SFH plays important role in reducing the symptoms of diabetes. In addition, SFH-treated db/db mice exhibited improved glucose tolerance with increased plasma insulin levels. Immunohistochemical and morphological analyses showed that SFH up-regulated insulin production by increasing pancreatic beta cell mass in the mice. In summary, our results suggest that SFH exerts anti-diabetic effects by increasing pancreatic beta cell mass in a non-insulin dependent diabetes mellitus mouse model.

Anti-hyperlipidemic effect of soybean extract fermented by Bacillus subtilis MORI in db/db mice / 한국실험동물학회지

The purpose of this study was to investigate the anti-hyperlipidemic effect of soy bean extract solution fermented by Bacillus subtilis MORI (BTD-1E) in obese db/db mice. Eight-week-old male db/db mice were administered 33.3 mg/kg BTD-1E solution orally once a day for four weeks. The BTD-1E group showed significantly lower body weight compared with the db control group (P<0.05). The BTD-1E group showed significantly lower serum total cholesterol and LDL cholesterol levels compared with the db control group, respectively (P<0.05, P<0.01). The BTD-1E group showed significantly decreased liver weight relative to final body weight compared with the db control group (P<0.01). After four weeks of BTD-1E administration, lipid droplets in the liver were apparently decreased in the BTD-1E group compared to the db control group. In summary, our results suggest that BTD-1E has an anti-hyperlipidemic effect in the obese mouse model.

Mapping of the Faded (fe) Gene to a Region between D10mit191 and D10mit44 on Mouse Chromosome 10 / 한국실험동물학회지

The faded mouse is a coat color mutant that shows faded coat color and age-related loss of pigmentation. This mutation is transmitted by an autosomal recessive gene with 100% penetrance. In the present study, we carried out linkage analysis of the faded (fe) gene using intra-specific backcross panels. Affected faded mice were carefully confirmed by their faded coat color at about 4 weeks of age. In the intra-specific backcross between faded and CBA mice (n=198), the fe gene was mapped to a region 2.1 cM distal to D10mit191. Therefore, the gene order was defined as follows: centromere-D10mit51 (12.4+/-2.4 cM)-D10mit191 (2.1+/-1.0 cM)-fe-D10mit44 (13.3+/-2.4 cM)-D10mit42 (14.4+/-2.5 cM). This linkage map of the fe locus will provide a good entry point to isolate the fe gene. Since the faded mouse has pigmentary abnormalities, this mutant may be a useful model for studies of pigmentary abnormalities in humans.

Subcellular localization of the transmembrane inner ear (Tmie) protein in a stable Tmie-expressing cell line / 한국실험동물학회지

Mutations in the transmembrane inner ear (Tmie) gene, which encodes the Tmie protein, have been attributed to deafness autosomal recessive 6 (DFNB6), an autosomal nonsyndromic recessive hearing loss disorder. Although the Tmie gene was identified a few years ago, little is known about subcellular localization of the Tmie protein. In order to address this, we developed a stable cell line expressing Tmie protein. The expression of Myc-tagged Tmie protein was confirmed by Western blot analysis using an anti-Myc antibody and localization of the Tmie protein was confirmed by immunostaining, using the anti-Myc antibody as well as the anti-tmie antibody. Our study demonstrates that the Tmie protein is localized mostly in the cellular membrane and to a lesser extent in cytoplasm. These results suggest that our Tmie expressing stable cell line provides a suitable in vitro model to explore Tmie synthesis and functions.

Animal Models of Periventricular Leukomalacia / 한국실험동물학회지

Periventricular leukomalacia, specifically characterized as white matter injury, in neonates is strongly associated with the damage of pre-myelinating oligodendrocytes. Clinical data suggest that hypoxia-ischemia during delivery and intrauterine or neonatal infection-inflammation are important factors in the etiology of periventricular leukomalacia including cerebral palsy, a serious case exhibiting neurobehavioral deficits of periventricular leukomalacia. In order to explore the pathophysiological mechanisms of white matter injury and to better understand how infectious agents may affect the vulnerability of the immature brain to injury, novel animal models have been developed using hypoperfusion, microbes or bacterial products (lipopolysaccharide) and excitotoxins. Such efforts have developed rat models that produce predominantly white matter lesions by adopting combined hypoxia-ischemia technique on postnatal days 1-7, in which unilateral or bilateral carotid arteries of animals are occluded (ischemia) followed by 1-2 hour exposure to 6-8% oxygen environment (hypoxia). Furthermore, low doses of lipopolysaccharide that by themselves have no adverse-effects in 7-day-old rats, dramatically increase brain injury to hypoxic-ischemic challenge, implying that inflammation sensitizes the immature central nervous system. Therefore, among numerous models of periventricular leukomalacia, combination of hypoxia-ischemia-lipopolysaccharide might be one of the most-acceptable rodent models to induce extensive white matter injury and ensuing neurobehavioral deficits for the evaluation of candidate therapeutics.

New Splicing Variants of the Murine Damaged DNA Binding 2 / 한국실험동물학회지

Damaged DNA binding (DDB) protein is an important gene in the repair of damaged DNA. DDB is a heterodimer (DDB1 and DDB2) protein, murine DDB2 has 10 exons about 1.5kb in size (Genbank Accession No. AY027937). Here we identified five DDB2 variants (M1-M5) from various mouse tissues that are generated by alternative splicing. We used reverse transcription-PCR (RT-PCR) to identify splicing variants and isolated PCR products using an agarose-gel PCR purification kit. All isolated PCR products were cloned and the structure of splicing variants was confirmed by sequencing. The first splicing variant M1 was generated by omission of exon 4. The second splicing variant M2, by omission of exons 4-5. The third variants M3 was generated by omission from the middle of exon 1 to exon 6 and was expressed in the heart. Fourth variants M4 was generated by omission of exon 2 and exons 4-7. M5, the last splicing variant was generated by omission of exons 4-7. M4 and M5 were expressed in the spleen. Analysis of tissue distribution by RT-PCR indicates that M1 is most highly expressed in the mouse brain. These results indicated that murine DDB2 has five splicing variants and splicing variants expression patterns were different depending on mouse tissue. Further functional studies of each splicing variants will provide more information about the molecular mechanism of DDB2 function and DDB2 gene expression regulation.

Establishment of a Research and Assessment System Using High Quality Non-Human Primates / 한국실험동물학회지

At present, eight non-human primate research facilities exist in Korea to examine the validity and safety of new bio-products, and to generate model animal systems using primates of low health status (low quality primates). However specific-pathogen free (SPF) primates (high quality primates) are the preferred choice for emerging disease studies and for numerous other research areas, including cell/gene therapy, stem cell research, regenerative studies, and brain science. Although international primate centers in developed countries have utilized high quality primate resources for many years, there has been little or no collaboration with less developed countries on primate research. Due to this, the establishment of a high quality primate research capacity is a core priority for the advancement of the biomedical research field in less developed countries. In this study, we investigated the demand for, and opportunities to support the development of this research capability.

Genetic Polymorphism of Jeju Horses by Microsatellite DNA Markers in Korea / 한국실험동물학회지

We determined the genetic diversity and distance between Jeju and Thoroughbred horses by genotyping for 20 microsatellite loci consisting of (TG)n repetitive sequence. The expected heterozygosity ranged from 0.1 to 0.789 in the Jeju horses and from 0.505 to 0.824 in the Thoroughbred horses. Polymorphic information content (PIC) values ranged from 0.09 to 0.709 in the Jeju horses and 0.365 to 0.730 in Thoroughbred horses. There were no significant differences in heterozygosity and PIC values between Jeju and Thoroughbred horsesHowever, LEX035 was estimated relatively high heterozygosity (0.789) and PIC value 0.709) in Jeju horses and LEX050 was respectively 0.824, and 0.730 in the Thoroughbred horses. We may conclude that the genetic differentiation was low between Jeju and Thoroughbred horses. LEX 050, LEX055, LEX059 and LEX 063 could be used as geneticmarkers for differentiating Jeju from Thoroughbred horses.

Assessment of experimental saccular aneurysm using selective angiography in common carotid artery of rabbits / 대한해부학회지

In order to study the treatment of aneurysms, the technique of making experimental aneurysms in laboratory animals must be established. In our study, to examine the feasibility of making experimental aneurysm and selective angiography on the common carotid artery in rabbits and to determine the size of experimental aneurysm after surgery, saccular aneurysms were fashioned on the right common carotid artery in 17 rabbits using a vein pouch technique. Selective angiography of the common carotid artery was performed immediately after surgery, and at 1 week, 4 weeks, and 8 weeks after surgery. Also, histological changes in the aneurysms were observed. In 16 rabbits with established successful experimental aneurysm, no differences were found in diet intake and behavior before and after surgery. The patency of the carotid artery was confirmed by selective angiography. The average size of the aneurysm immediately after surgery was similar to that of 1 week postoperatively in selective angiography, however it increased with time at 4weeks and 8 weeks. Histologically, infiltration of inflammatory cells and hemorrhage were found at the junction of the carotid artery and the vein pouch at 1 week, which disappeared at 4 weeks and 8 weeks. This study suggests experimental saccular aneurysm using the vein pouch technique might form aneurysms similar to that of the human in its properties such as increment of size, and selective angiography might be suitable for assessment of experimental aneurysm. Therefore, this animal model may be suitable for investigating new treatment methodologies for human aneurysms.

Immunolocalization of anion exchanger 1 (Band 3) in the renal collecting duct of the common marmoset

The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/ non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl-/HCO3- exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.