• 한국수의병리학회지
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• 제1권2호
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• pp.119-124
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• 1997

The objective of this study was to develop digoxigenin (DIG)-labeled in situ hybridization (ISH) test for diagnosis of Aujeszky's Disease(AD) in infected organs. Specific DNA with well conserved gene sequences encoding gp50 antigen in AD virus (ADV) was obtained by Polymerase Chain Reaction (PCR) method. A pair of oligonucleotide primers used in PCR allowed amplification of a 217 bp sequence from the gp50 ADV gene. The DNA was then labeled with DIG by primer labeling method for use as probe in ISH test to detect ADV nucleic acids in various tissue. Positive hybridization was demonstrated by dark pigmentation in nuclei and cytoplasm of ADV infected cells particularly in brain tonsillar crypt epithelium and pulmonary alveolar cells. This result suggests that ISH is a valuable sensitive and rapid diagnostic test for AD.

• 대한수의학회지
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• 제36권4호
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• pp.845-858
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• 1996

The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs.

• International Journal of Oral Biology
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• 제41권2호
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• pp.97-103
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• 2016

Mammals have 3 pairs of major salivary glands i.e., the parotid, submandibular, and sublingual glands. Saliva secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and ductal cells. Primary saliva is secreted by acinar cells and modified during ductal flow. Recently, of the murine 35 bitter taste receptors, Tas2r108 was expressed at highest levels in the submandibular gland by qPCR. Further, Tas2r108-transfected cells respond to a range of bitter compounds, such as denatonium, quinine, colchicine, diphenidol, caffeine and dapson. The objective of the present study was to characterize the expression of Tas2r108 mRNA in acinar and/or ductal cells of the submandibular gland using in situ hybridization (ISH). Male 42-60 days old DBA2 mice were used in the study. Messenger RNAs were extracted from the submandibular gland for generating digoxigenin (DIG) labeled-cRNA probes. These probes were transcribed in anti-sense and sense orientation using T7 RNA polymerase. Dot blot hybridization was performed using DIG labeled-cRNA probes, in order to estimate integrity and optimal diluting concentration of these probes. Subsequently, ISH was performed on murine submandibular gland to detect Tas2r108 mRNA. Dot blot hybridization data demonstrated that Tas2r108 DIG labeled-cRNA anti-sense probes specifically detected Tas2r108 cDNA. ISH results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular gland, whereas no staining was visible in sense controls. Interestingly, the Tas2r108 expression levels were higher in acinar than ductal cells. These results suggested that Tas2r108 might be more associated with primary saliva secretion than with ductal modification of saliva composition.

• Fisheries and Aquatic Sciences
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• 제9권4호
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• pp.146-152
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• 2006

Systemic infections of maricultured fishes by Megalocytivirus species have occurred over a broad area in South Korea, causing extensive economic loss. We developed digoxigenin-labeled nucleic acid probes against the 230-bp ATPase and 311-bp major capsid protein (MCP) of rock bream Oplegnathus fasciatus iridovirus (RBIV) using polymerase chain reaction, and an in situ hybridization (ISH) method to detect Megalocytivirus in formalin-fixed tissues of mariculture species (rock bream, sea bass, and olive flounder). ISH-positive cells were abundant in the hematopoietic and connective tissues of various organs, while brain tissue showed little or no signal. The ISH procedure can become an important diagnostic tool in complement with histopathological methods, and advances epidemiological studies on the origin and distribution of Megalocytivirus in mariculture.

• Asian-Australasian Journal of Animal Sciences
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• 제23권4호
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• pp.530-536
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• 2010

Cdc25B is a mitotic regulator that might act as a starter phosphatase to initiate the positive feedback loop at the entry into mitotic (M) phase. In the present study, distribution of Cdc25B mRNA in duodenal mucosa of the chicken was demonstrated by means of in situ hybridization histochemistry (ISHH) using sense and antisense digoxigenin (DIG)-labeled RNA probes. The results showed that there were many labeled cells distributing in the duodenal mucosa of the adult chicken. Of these labeled cells, 81.60${\pm}$9.63% of Cdc25B mRNA positive cells was distributed in the basilar part and mid-portion of the intestinal gland and 36.21${\pm}$8.81% in the middle and basilar portion of villi of the small intestine of the chicken, respectively. Most of these labeled cells were positive in the regions of the stem cell and proliferation. The signals of ISHH decreased from basilar to upper part in the crypt of Lieberkuhn and weakened in the inferior villi of the duodenum. Moreover, the positive signals were both in the cytoplasm and cell nucleus. However, the labeled cells were negative in both the lamina muscularis mucosae and muscular layer. The results of ISHH suggested the existence of Cdc25B mRNA and vigorous proliferation activities in the duodenal mucosa of adult chicken, replenishing the cells which had sloughed off from the superior part of the villus. Our results provide some molecular evidence for a regular pattern of avian intestinal epitheliosis and functional partition and provide an approach to further study of the locations of Cdc25B in the chicken.

• 대한수의학회지
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• 제36권4호
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• pp.859-871
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• 1996

This study was conducted to elucidate the distribution of Aujeszky's disease viral nucleic acids and antigens in the central nervous system (CNS) of piglets. The first Korean isolate of Aujeszky's disease virus(ADV) that isolated from naturally infected piglets in Yang San, was inoculated into 32 day old piglets with $10^{5.9}TCID_{50}/ml$ through intranasal or intramuscular route. These piglets were sacrificed at every 24hrs for 8 days. The immunohistochemistry (IHC) was conducted to detect the viral antigens in paraffin-embedded tissue sections using avidin-biotin-peroxidase complex (ABC) method. The viral nucleic acids were detected by in situ hybridization (ISH) using ADV specific DNA probe labeled with digoxigenin. The ADV antigens were detected in reticuloendothelial cells of spleen, lymph nodes and tonsil, alveolar walls, leptomeningeal vascular walls, inflammatory foci of each organ, and nerve cells. The viral nucleic acids were detected in the spinal trigeminal nucleus and its tracts of the pons and medulla oblongata by the ISH technique. The pathways of AD viruses in CNS were determined by IHC and ISH. In the intranasally inoculated group, the viruses in nasal mucosa moved to medulla oblongata and pons through the trigeminal nerve. In case of intramuscullarly inoculated group, viruses moved to brain via lymphoid organs or spinal nerves from sciatic nerves.

• Archives of Pharmacal Research
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• 제22권3호
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• pp.243-248
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• 1999

Sialic acids are key determinants for biological processes, such as cell-cell interaction and differentiation. Sialyltransferases contribute to the diversity in carbohydrate structure through their attachment of sialic acid in various terminal positions on glycolipid and glycoprotein (N-linked and O-linked) carbohydrate groups. Gal$\beta$ 1,3(4)GlcNAc $\alpha$2,3-sialyltransferase (ST3Gal III) is involved in the biosynthesis of $sLe^{X}$ and sLe^{a}$ known as selection ligands and tumor-associated carbohydrate structures. The appearance and differential distribution of ST3Gal III mRNA during mice embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. On E9, all tissues were positive for ST3Gal III mRNA expression whereas ST3Gal III mRNA on E11 was not detected throughout all tissues. On E13, ST3GAl III mRNA was expressed in different manner in various tissues. In this stage, ST3Gal III mRNA was positive only in the liver, pancreas and bladder. On E15, specific signal for ST3GAl III was detected in the liver, lung and forebrain. These results indicate that ST3Gal III is differently expressed at developmental stages of mice embryo, and this may be importantly related with regulation of organogenesis in mice.

• Archives of Pharmacal Research
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• 제23권5호
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• pp.525-530
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• 2000

mST3GaIV synthesizes ganglioside GM3, the precursor for simple and complex a- and b- series gangliosides, and the expression and regulation of mST3GaIV (CMP-NeuAc: lactosylceramide $\alpha$2,3-sialyltransferase) activity is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in variety of cellular processes such as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell-cell interactions. To understand the developmental expression of mST3GaIV in mice, we investigated the spatial and temporal expression of mST3GaIV mRNA during the mouse embryogenesis [embryonic (E) days; 19, E11, E13, E15] by in situ hybridization with digoxigenin-labeled RNA probes. All tissues from 19 and E11 were positive for mST3GaIV mRNA. On E13, mST3GaIV mRNA was expressed in various neural and non-neural tissues. In contrast to these, on E15, the telencephalon and liver produced a strong expression of mST3GaIV which was a quite similar to that of E13. In this stage, mST3GaIV mRNA was also expressed in some non-neural tissues. These data indicate that mST3GaIV is differently expressed at developmental stages of embryo, and this may be importantly related with regulation of organogenesis in mice.

• Asian-Australasian Journal of Animal Sciences
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• 제15권11호
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• pp.1531-1535
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• 2002

It has been known that the sex of chicken cells can be most accurately identified by fluorescence in situ hybridization (FISH). However, the presently available FISH has not been widely used for sex identification, because the procedures for cell preparation and FISH itself are complicated and time-consuming. The present study was undertaken to test a rapid FISH procedure for sexing chicken. A FISH probe was simultaneously synthesized and labeled with digoxigenin by polymerase chain reaction (PCR) targeting a 416 bp segment of the 717 bp XhoI family fragment which is repeated over 10 thousand times exclusively in the W chromosome. Sexing by FISH was performed on cytological preparations of early embryos, adult lymphocytes and feather pulps of newly hatched chicks. The DNA probe hybridized to all types of uncultured interphase as well as metaphase female but not male cells that had been examined. Moreover, consistent with the known site of the XhoI family, the hybridization signal was localized to the pericentromeric region of the W chromosome. We, therefore, conclude that the present PCR-based FISH can be used as a rapid and reliable sex identification procedure for chicken.

• Asian-Australasian Journal of Animal Sciences
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• 제22권6호
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• pp.781-787
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• 2009

The sense and antisense digoxigenin-labeled RNA probes of four genes, Cdc25A, Cdc25B, Sox2 and Mnb, were produced by using SP6 and T7 RNA polymerases, respectively, and in vitro transcription. Expression patterns of the four genes were detected by in situ hybridization in HH (Hamburger and Hamilton) stage 10 chick embryos. In general, expression patterns of the four genes were similar. mRNA of the four genes was mostly restricted to the entire CNS (central nervous system). All were confined to an identical region, neural tube, neural groove and caudal neural plate, corresponding to the notochord or spinal cord, but there was some distinction in specific region or in concentration, for example in somites. The overlap in expression at the same developmental stage in the CNS suggests that the four genes may be functional similar or related in CNS development. Expression patterns of the four genes support specific roles of these regulators in the developing CNS.