• Biomedical Science Letters
• /
• v.26 no.2
• /
• pp.114-119
• /
• 2020

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

• Journal of Biomedical Engineering Research
• /
• v.42 no.3
• /
• pp.125-142
• /
• 2021

The POCT (point-of-care test) sensing that has been a fast-developing field is expected to be a next generation technology in health care. The POCT sensors for the detection of proteins, small molecules and especially nucleic acids have lately attracted considerable attention. According to the World Health Organization (WHO), the POCT methods are required to follow the ASSURED guidelines (Affordable, Sensitive, Specific, User- friendly, Robust and rapid, Equipment-free, Deliverable to all people who need the test). Recently, several CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic techniques using the sensitive gene recognition function of CRISPR have been reported. CRISPR/Cas (Cas, CRISPR associated protein) systems based detection technology is the most innovative gene analysis technology that is following the ASSURED guidelines. It is being re-emerged as a powerful diagnostic tool that can detect nucleic acids due to its characteristics that enable rapid, sensitive and specific analyses of nucleic acid. The first CRISPR-based diagnosis begins with the discovery of the additional function of Cas13a. The enzymatic cleavage occurs when the conjugate of Cas protein and CRISPR RNA (crRNA) detect a specific complementary sequence of the target sequence. Enzymatic cleavage occurs on not only the target sequence, but also all surrounding non-target single-stranded RNAs. This discovery was immediately utilized as a biosensor, and numerous sensor studies using CRISPR have been reported since then. In this review, the concept of CRISPR, the characteristics of the Cas protein required for CRISPR diagnosis, the current research trends of CRISPR diagnostic technology, and some aspects to be improved in the future are covered.

• Fisheries and Aquatic Sciences
• /
• v.13 no.2
• /
• pp.112-117
• /
• 2010

Altered mRNA expression of two metallothionein isoforms (MT-IA and MT-IB) in response to acute heavy metal exposure was examined in mud loach, Misgurnus mizolepis, fry using a real-time RTPCR assay. Sublethal exposure (1 or 5 ${\mu}M$) to Cd, Cr, Fe, Mn, Ni, and Zn resulted in highly variable transcriptional responses of the two MT isoforms to the heavy metal ions, including upregulation, a steady state, and downregulation. Overall, the most potent inducer of both MT isoforms was Cd (up to 6-fold). Another exposure experiment using a series of doses of Cu revealed that the stimulation patterns of the two MT isoforms differed: MT-IA transcription was soon saturated at higher concentrations (about 2-fold at 1-4 ${\mu}M$ of Cu), whereas the activation of MT-IB was more dependent on the treatment dose (increased up to 5-fold at 3 ${\mu}M$). The isoform-specific allotment of constitutive and inducible functions was not as clear in fry as in adult tissues. Coordinated interaction between the MT-IA and MT-IB isoforms was hypothesized based on the finding that MT-IA represented a primary action under 'less stressful' or 'sublethal' conditions, whereas the activation of MT-IB became important under 'more stressful' or 'lethal' circumstances in this species.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2019.04a
• /
• pp.117-117
• /
• 2019

Kirengeshoma koreana Nakai (K. koreana)was Saxifragaceae and rare plants in Korea, which is classified as an Critically Endangered (CR) species in Korea. Therefore, most of the studies on it were ecological and taxonomic, and there are no studies on biological activity. In this study, we evaluated the whitening activity of K. koreana extract (KKE). Melanogenesis Inhibitory effects were demonstrated by western-bot and RT-PCR for the effects of KKE on MITF, tyrosinase, TRP-1 and TRP-2 in IBMX-treated B16F10 melanoma cells. IBMX were reported as melanin synthesis enhancers. It could increase intracellular melanin synthesis by activation of the microphthalmia-associated transcription factor (MITF) signaling pathway. KKE showed no cytotoxicity at B16F10. In addition, KKE effectively inhibited the protein and mRNA levels of MITF, tyrosinase, TRP-1 and TRP-2. In conclusion, KKE inhibited melanin synthesis by inhibiting the expression of MITF and its downstream pathways tyrosinase, TRP-1 and TRP-2. Therefore, it was confirmed that K. koreana is a valuable resource for functional cosmetic and biomaterials.

• Journal of Acupuncture Research
• /
• v.19 no.6
• /
• pp.111-124
• /
• 2002

A line of study reported that electroacupuncture(EA) modulate natural killer cell(NK Cell) activities. One report suggested that EA enhanced splenic interferon-gamma($IFN-{\gamma}$), interleukin-2(IL-2), and NK cell activity in Sprague-Dawley rats. Another study suggested that $IFN-{\gamma}$ mediates the up-regulation of NK cell activity, and endogenous ${\beta}$-endorphin secretion also play a role in the up-regulation of NK cell activity induced by EA stimulation. In order to better understand the molecular regulation underlying the activation of NK cell induced by EA, we have utilized cDNA microarray to elucidate how EA alters program of gene expression of spleen in rats. First, we divided three groups, group I was EA group treated with EA in restriction holder, group II was sham group with only holder stress, and last group III was control group with no treatment. We measured NK cell activity after EA stimulation three times for 2 days using $^{51}Cr$ release assay. Second, Biotin-labeled cDNA probes synthesized from EA group and sham group, were competitively hybridized to the microarray that contained variable genes. Such high-throughput screening has identified a number of EA-responsive gene candidates. Of these, we found that EA induced a subset of genes of genes that functionally could modulatory effects on NK cell activity. Genes(vascular cell adhesion molecule-1, protein-tyrosine kinase, CD94 mRNA) related to boost NK cell activity, were increased by EA And, genes(protein-tyrosine-phospatase mRNA, protein-tyrosine phosphatase(SHP-1) mRNA) related to inhibit NK cell activity, were decreased by EA. These EA-responsive genes may provide key insights from which to understand mechanisms of activation of NK cell induced by EA.

• Journal of Marine Life Science
• /
• v.4 no.1
• /
• pp.38-43
• /
• 2019

The deformity occurring at the early developmental stage of red spotted grouper (Epinephelus akaara) causes detrimental effects on the process of juvenile production. In this study, we have compared the expressions of several key genes (insulin like growth factor 1: IGF-1, bone morphogenic protein 4: BMP4, peroxisome proliferator-activated receptors γ: PPARγ, matrix Gla protein: MGP) for morphogenesis between normal and 2 types (cephalic and jaw) of deformed juvenile fish. Expression of these genes were investigated in the brain, liver and muscle of each group of fish (n=20) by real-time PCR. Expression of IGF-1 and BMP4 mRNA in the brain and liver showed significant difference between normal and deformed fish (p<0.05). However, no difference was observed in the expression of PPARγ and MGP mRNA between normal and deformed fish in any tissues. It seems certain that IGF-1 and BMP4 are associated with the state of deformity in juvenile red spotted grouper.

• Journal of Food Hygiene and Safety
• /
• v.29 no.1
• /
• pp.31-39
• /
• 2014

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.84-84
• /
• 2003

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. DNA methylation is a highly plastic and critical component of mammalian development The DNA methyltransferases (Dnmts) are responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. The maintenance DNA methyltransferase enzyme, Dnmt 1, and the de novo methyltransferase, Dnmt3a and Dnmt3b, are indispensable for development because mice homozygous for the targeted disruption of any of these genes are not viable. The occurrence of DNA methylation is not random, and it can result in gene silencing The mechanisms underlying these processes are poorly understood. It is well established that DNA methylation and histone deacetylation operate along a common mechanistic pathway to repress transcription through the action of methyl-binding domain proteins (MBDs), which are components of, or recruit, histone deacetylase (HDAC) complexes to methylated DNA. As a basis for future studies on the role of the DNA-methyl-transferase in porcine development, we have isolated and characterized a partial cDNA coding for the porcine Dnmt1. Total RNA of testis, lung and ovary was isolated with TRlzol according to the manufacture's specifications. 5 ug of total RNA was reverse transcribed with Super Script II in the presence of porcine Dnmt 1 specific primers. Standard PCRs were performed in a total volume of 50 ul with cDNA as template. Two DNA fragmenets in different position were produced about 700bp, 1500bp and were cloned into pCR II-TOPO according to the manufacture's specification. Assembly of all sequences resulted in a cDNA from 158bp of 5'to 4861bp of 3'compare with the known human maintenance methyltransferase. Now, we are cloning the unknown Dnmt 1 region by 5'-RACE method and expression of Dnmt 1 in tissues from adult porcine animals.

• IMMUNE NETWORK
• /
• v.9 no.6
• /
• pp.255-264
• /
• 2009

Background: Chronic low grade inflammation is closely linked to type II diabetes, obesity, and atherosclerosis. Macrophages play a key role in the regulation of pro- or anti-inflammatory actions at the lesion sites of disease. Components of cordyceps militaris, cordycepin and adenosine, have been used for the modulation of inflammatory diseases. The effects of cordycepin in the modulation of macrophages have yet to be be elucidated. We investigated the effects of cordycepin and adenosine on the morphological changes of macrophages under the inflammatory condition of LPS and an anti-inflammatory condition involving high concentrations of adenosine. Methods: We confirmed the mRNA levels of the M1/M2 cytokine genes through RT-PCR and morphological change. Results: LPS-activated macrophages returned to their inactivated original shape, i.e., they looked like naive macrophages, through the treatment with high concentrations of cordycepin ($40{\mu}g/ml$). LPS and adenosine activated macrophages also returned to their original inactivated shapes after cordycepin treatment; however, at relatively higher levels of cordycepin than adenosine. This change did not occur with relatively low concentrations of cordycepin. Adenosine down-regulated the gene expression of M1 cytokines (IL-$1{\beta}$, TNF-${\alpha}$) and chemokines (CX3CR1, RANTES), such as cordycepin. Additionally, M2 cytokines (IL-10, IL-1ra, TGF-${\beta}$) were up-regulated by both cordycepin and adenosine. Conclusion: Based on these observations, both cordycepin and adenosine regulated the phenotypic switch on macrophages and suggested that cordycepin and adenosine may potentially be used as immunomodulatory agents in the treatment of inflammatory disease.

• The Journal of Internal Korean Medicine
• /
• v.28 no.4
• /
• pp.681-693
• /
• 2007

Background and Objective : Atractylodes japonica (AJ) is a commonly-used herbal medicine in Asian countries such as Korea, China and Japan. The present study was designated to evaluate the direct effects of AJ on helper T cell activities and on Th1/Th2 lineage development in vitro. Materials and Methods : Spleen cells from 8-week BALB/c mice were cultured in CR extracts containing medium without activation for 24 hours and with activation for 48 hours. CD4+ T cells were isolated and analyzed for mRNA expression levels of INF-$\gamma$, IL-4, T-bet and GATA-3 by RT-PCR and secretion cytokines levels of INF-$\gamma$, IL-2, IL-4, IL-5 and IL-10 by ELISA. Results : The results demonstrated that AJ had no mitogenic effects on unstimulated CD4+ T cells, but augmented CD4+T-cell proliferation upon activation with anti-CD3/anti-CD28 antibodies in a dose-dependent manner. AJ treatment significantly increased CD4+ T cell population and IFN-$\gamma$ expression was significantly enhanced, while IL-4 expression significantly decreased. In addition, in vitro Th1/Th2 polarization experiments revealed that AJ enhanced IFN-$\gamma$ secretion in Th1 cells, but reduced the IL-4 in Th2 cells in dose-dependent manner. Conclusion : These results suggest that AJ treatment could be a desirable alternative therapy for the prevention or correction of Th2 dominant pathological disorders, such as allergy and asthma.