• Journal of Veterinary Clinics
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• v.27 no.5
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• pp.508-513
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• 2010

Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

• Journal of Aquaculture
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• v.13 no.1
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• pp.79-85
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• 2000

The effects of estrogen and androgens on Vg gene expression were examined in primary hepatocyte culture and livers of the immature male trout. Specific primers of Vg cDNA were designed with already reported Vg gene nucleotide sequences. PCR product was sequenced and verified with Vg cDNA of rainbow trout. Total RNA was extracted from the cultured hepatocytes and livers of steroid-treated rainbow trout and then it was analyzed by reverse transcriptase- polymerase chain reaction (RT-PCR) analysis. The Vg mRNA and Vg protein synthesis were increased in rainbow trout in vivo and in vitro with E$_2$ and methyltestosterone (MT) There were dose and time-related effects of E$_2$ and MT on vitellogesis. Androgens such as progesterone androsterone and testosterone also stimulated Vg mRNA expression in vitro. The results show that androgens as well as E$_2$ can induce expression of Vg mRNA in trout in vivo and in vitro.

• Journal of fish pathology
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• v.10 no.1
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• pp.39-44
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• 1997

Metallothionein is an important and essential protein to control the intracellular concentration of heavy metals, which exist in all organisms from bacteria to vertebrates. Although the detailed functions and induction mechanisms of metallothionein gene have not been clearly characterized until yet, the structure of several metallothionein genes has been revealed. Especially, piscine metallothionein is regarded as an important protein because it is induced by several heavy metal pollutants and environmental stress and it could be determined the comparative amount of heavy metals and the extent of environmental stress by assaying the RNA transcript of metallothionein gene in the method of the quantitative RT-PCR(Reverse Transcriptase Polymerase Chain Reaction). In this study I have characterized the 450 bp PCR fragment of metallothionein gene amplified by using the mixture of internal specific primers and universal 3' end primer. The nucleotide sequence analysis of 450 bp PCR fragment amplified in cDNA library of channel catfish did not show strong homology to other piscine metallothionein genes.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.4
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• pp.153-157
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• 2009

The production of extended-spectrum ${\beta}$-lactamases ($ESBL_S$) of the TEM or SHV type by bacterial pathogens is a major threat to the use of the clinically important expanded-spectrum cephalosporins. The characterization of the SHV ESBLs producing Klebsiella pneumoniae strains present in clinical isolates is time-consuming processes. We describe here in the development of a novel system, which consists of a real time PCR. We found 11 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disk synergy test showed 8 ESBL positive and conventional PCR showed 10 SHV ESBL positive, which were K. pneumoniae strains isolates. By real time PCR analysis, SHV gene in 11 of 11 strains were identified. When sequencing analysis was compared with real time PCR, both analysis were presented 99% similarity. In this study, we used a rapid, sensitive, and specific real-time PCR (RT-PCR) method for detection of the assay SHV ESBL producing K. pneumoniae strains in clinical isolates.

• Journal of the korean academy of Pediatric Dentistry
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• v.37 no.3
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• pp.275-287
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• 2010

Deep caries may induce pulpitis and the pulpal tissue interacts with microbial invasion. The immune response to protect the pulpal tissue can be mediated by cellular signal molecules produced by the pulpal cells. The understanding of these processes is important to find future therapeutic method for the diseased pulp. The pulp tissue from sound teeth was set as control group (n=30) and the pulp tissue from decayed teeth was set as test group (n=30). Total RNA was extracted from the pulp of each group and it was used for cDNA microarray and reverse transcriptase-polymerase chain reaction(RT-PCR). The expression of TGF-${\beta}1$ was studied by immunohistochemistry. The results were as follows: 1. cDNA microarray analysis identified 520 genes with 6-fold or greater difference in expression level with 143 genes more abundant in health and 377 genes more abundant in disease. 2. The RT-PCR analysis was done for randomly selected 14 genes and the results supported the result of cDNA microarray assay. 3. TGF-${\beta}1$ was highly expressed in the carious pulp and it was found in odontoblast by immunohistochemistry. In conclusion, many cytokines were found to be significantly changed their expression in the diseased pulp(/M/>1.6).

• Korean Journal of Plant Tissue Culture
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• v.27 no.1
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• pp.7-12
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• 2000

Explants of lettuce (Lactuca sativa L.) were co-cultivated with Agrobacterium tumifacience GV 3101 strain containing nptII gene and cold regulated gene (BN115) from Brassica napus for transformation. Multiple shoots were obtained from the explants in the selection medium (MS basal medium supplemented with 100 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA, 0.5 mg/L kinetin) after 3 to 4 weeks of co-culture. The putative transgenic shoots were transferred to rooting medium (1/2 MS basal medium supplemented with 100 mg/L kanamycin and 250 mg/L carbenicillin). The selected shoots were tested with PCR analysis using nptll, BN115 primers whether cold-regulated gene was introduced to genome of the plants. The vir G primers were particularly used to check contamination of Agrobacterium during PCR analysis. The nptII and BN115 primers produced the specific PCR bands in the putative transgenic lines but the vir G primers did not. These results confirmed that the PCR products were not the result of contamination with Agrobacterium. Additionally the Southern analysis of the PCR products and RT-PCR analysis proved that the cold-regulated gene was successfully integrated and transcribed in the putative transgenic lettuce plants.

• Journal of Life Science
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• v.13 no.1
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• pp.105-109
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• 2003

Epoxide hydrolase (EH) gene from Aspergillus niger #33 was cloned from cDNA library generated by reverse transcriptase-polymerase chain reaction (RT-PCR). The nucleotide sequence analysis revealed that the deduced amino acid sequence was almost similiar to that of A. niger LCP521 previously reported. The cloned EH gene was transformed into Saccharomyces cerevisiae and expressed by addition of galactose. The recombinant S. cerevisiae showed hydrolytic activity toward racemic phenyl oxirane substrate based on chiral GC analysis, and can be used as a potential biocatalyst for the preparation of chiral phenyl oxirane.

• Korean Journal of Veterinary Research
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• v.41 no.3
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• pp.333-342
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• 2001

To detect the genetic variations among infectious bursal disease (IBD) vaccine strains, the hypervariable region of VP2 gene of seven IBDV vaccine strains were amplified using reverse transcriptase/polymerase chain reation(RT/PCR). Ampllified PCR products of IBDV were cloned, sequenced, and compared with published sequences for IBDV. Vaccine strains (JOONG, HAN, B7, IB, BU2, G2, CIL) used in Korea and Korean field isolates (SH/92, K1, 310) had 81%(310 and HAN) ~ 98%(SH/92 and CIL) amino acid sequence similarity. Vaccine strains had 80%(HAN and IB) ~ 99%(JOONG and BU2) amino acid sequence similartiy. Intermediate plus vaccine strain, CIL was not substituted at positions 279(D $\rightarrow$ N) and 284(A $\rightarrow$ T), and conserved in serine-rich heptapeptide. At the two hydrophilic region, JOONG, IB and Bu2 strains had identical amino acid sequence comparing with STC strain. By phylogenetic analysis, JOONG and DAE strains were categorized in same group with BU2. The CIL and STC strains closely related but seperated from G2, HAN, B7 and IB strains.

• BMB Reports
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• v.29 no.2
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• pp.137-141
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• 1996

A partial cDNA encoding a Korean radish isoperoxidase was obtained from a cDNA library prepared from 9 day old radish root. In order to obtain Korean radish isoperoxidase cDNA, 5' RACE (rapid amplification cDNA end) PCR was performed and a cDNA (prxK1) encoding a complete structural protein was obtained by RT (reverse transcription)-PCR. Sequence analysis revealed that the length of the cDNA was 945 base pairs, and that of the mRNA transcript was ca. 1.6 kb. The deduced amino acid of the protein were composed of 315 amino acid residues and the protein was 92% homologous to turnip peroxidase, and 46% to 50% homologous to other known peroxidases. The 945 bp cDNA encoding Korean radish isoperoxidase was overexpressed in Escherichia coli up to approximately 9% of total cellular protein. The recombinant fusion protein exhibited 43 kDa on SDS-PAGE analysis and the activity level of the recombinant nonglycosylated protein was two fold higher in IPTG induced cell extracts than that of uninduced ones.

• The Plant Pathology Journal
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• v.16 no.1
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• pp.43-47
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• 2000

Citrus tristeza virus (CTV) is the causal agent of one of the most important diseases of citrus. Recently, CTV has been detected in Cheju Island by ELISA. The coat protein (CP) gene of CTV isolated form Cheju Island was cloned by RT-PCR and the nucleotide was analyzed in this study. Citrus leaves were collected from trees showing decline symptoms from various region of Cheju Island in the summer of 1998 and 1999. The CP gene open reading frame is composed of 670 nucleotides and encodes a polypeptide of 223 amono acids. Sequence analysis the CP gene revealed that two CTV strains present in Cheju Island. Viruses collected form Sogwipo area and Cheju City area in 1999 ahowed 91-93% nucleotide sequence homology with CTV T36 strain. Viruses collected form Cheju City area in 1999 and Sogwipo City in 1998 showed 94-98% nucleotide sequence homology with CTV SY568 strain. A efficient viral RNA extraction methods was developed by modifying procedure for animal virus RNA purification methods and PCR product was detected form one tenth of RNA purified from as small as 45 mg fresh or frozen tissue.