• Applied Biological Chemistry
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• 제39권4호
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• pp.260-264
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• 1996

Cymbidium속 난의 형질전환계를 확립하기 위해서, microprojectile bombardment 방법을 이용하여 춘란(Cymbidium virescence)의 rhizome 조직세포에 외래 유전자를 도입하는 조건을 검토하였다. 각 parameter별 적정조건으로서 텅스텐 입자의 크기는 $1.11\;{\mu}m$, He 가스 압력은 $77.33kg/cm^2$, gap distance는 6.35mm, target distance는 7.0cm 이었다. DNA피복입자를 투사한 $400{\mu}m$ 두께의 rhizome 절편을 2개월간 배양한 뒤 GUS 활성을 조사한 결과 이 유전자가 발현되는 세포들이 관찰되었다. Kanamycin (100 mg/L)을 첨가한 배지에서 6개월 동안 선택배양을 통해 얻은 rhizome의 DNA를 PCR로 분석한 결과 nptII 유전자의 삽입을 확인할 수 있었다.

• 대한의생명과학회지
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• 제12권2호
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• pp.81-89
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• 2006

The ischemia-responsive 94 gene (irp94) encoding a 94 kDa endoplasmic reticulum resident protein was investigated its molecular properties associated with unfoled protein responses. First, the expression of irp94 mRNA was tested after the reperfusion of the transient forebrain ischemia induction at the central nervous system in three Mongolian gerbils. Second, irp94 expression in PC12 cells, which are derived from transplantable rat pheochromocytoma cultured in the DMEM media, was tested at transcriptional and translational levels. The half life of irp94 mRNA was also determined In PC12 cells. Last, the changes of irp94 mRNA expression were investigated by the addition of various ER stress inducible chemicals (A23187, BFA, tunicamycin, DTT and $H_2O_2$) and proteasome inhibitors, and heat shock. High level expression of irp94 mRNA was detected after 3 hours reperfusion in the both sites of the cerebral cortex and hippocampus of the gerbil brain. The main regulation of irp94 mRNA expression in PC 12 cells was determined at the transcriptional level. The half life of irp94 mRNA in PC12 cells was approximately 5 hours after the initial translation. The remarkable expression of irp94 mRNA was detected by the treatment of tunicamycin, which blocks glycosylation of newly synthesized polypeptides, and $H_2O_2$, which induces apoptosis. When PC12 cells were treated with the cytosol proteasome inhibitors such as ALLN (N-acetyl-leucyl-norleucinal) and MG 132 (methylguanidine), irp94 mRNA expression was increased. These results indicate that expression of irp94 was induced by ER stress including oxidation condition and glycosylation blocking in proteins. Expression of irp94 was increased when the cells were chased after heat shock, suggesting that irp94 may be involved in recovery rather than protection against ER stresses. In addition, irp94 expression was remarkably increased when cytosol proteasomes were inhibited by ALLN and MG 132, suggesting that irp94 plays an important role for maintaining the ERAD (endoplasmic reticulum associated degradation) function.

• Journal of Plant Biotechnology
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• 제4권4호
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• pp.143-150
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• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• Journal of Plant Biotechnology
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• 제2권2호
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• BMB Reports
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• 제39권3호
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• pp.329-334
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• 2006

RNAi (RNA interference) has become a popular means of knocking down a specific gene in vivo. The most common approach involves the use of chemically synthesized short interfering RNAs (siRNAs), which are relatively easy and fast to use, but which are costly and have only transient effects. These limitations can be overcome by using short hairpin RNA (shRNA) expression vectors. However, current methods of generating shRNA expression vectors require either the synthesis of long (50-70 nt) costly oligonucleotides or multi-step processes. To overcome this drawback, we have developed a one-step short-oligonucleotides-based method with preparation costs of only 15% of those of the conventional methods used to obtain essentially the same DNA fragment encoding shRNA. Sequences containing 19 bases homologous to target genes were synthesized as 17- and 31-nt DNA oligonucleotides and used to construct shRNA expression vectors. Using these plasmids, we were able to effectively silence target genes. Because our method relies on the onestep ligation of short oligonucleotides, it is simple, less error-prone, and economical.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.449-455
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• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• International Journal of Oral Biology
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• 제45권1호
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• pp.25-31
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• 2020

Enamel knot (EK)-a signaling center-refers to a transient morphological structure comprising epithelial tissue. EK is believed to regulate tooth development in early organogenesis without its own cellular alterations, including proliferation and differentiation. EKs show a very simple but conserved structure and share functions with teeth of recently evolved vertebrates, suggesting conserved signaling in certain organs, such as functional teeth, through the course of evolution. In this study, we examined the expression patterns of key EK-specific genes including Dusp26, Fat4, Meis2, Sln, and Zpld1 during mice embryogenesis. Expression patterns of these genes may reveal putative differentiation mechanisms underlying tooth morphogenesis.

• Journal of Korean Neurosurgical Society
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• 제43권2호
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• pp.97-104
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• 2008

Objective: Transient receptor potential vanilloid subfamily type 1 (TRPV1), a most specific marker of the nociceptive primary afferent, is expressed in peptidergic and non-peptidergic primary afferents innervating skin and viscera. However, its expression in sensory fibers to skeletal muscle is not well known. In this study, we studied the neurochemical characteristics of TRPV1-positive primary afferents to skeletal muscles. Methods: Sprague-Dawley rats were injected with total $20{\mu}l$ of 1% fast blue (FB) into the gastrocnemius and erector spinae muscle and animals were perfused 4 days after injection. FB-positive cells were traced in the L4-L5 (for gastrocnemius muscle) and L2-L4 (for erector spinae muscle) dorsal root ganglia. The neurochemical characteristics of the muscle afferents were studied with multiple immunofluorescence with TRPV1, calcitonin gene-related peptide (CGRP) and $P2X_3$. To identify spinal neurons responding to noxious stimulus to the skeletal muscle, 10% acetic acids were injected into the gastrocnemius and erector spinae muscles and expression of phospho extracellular signal-regulated kinase (pERK) in spinal cords were identified with immunohistochemical method. Results: TRPVl was expressed in about 49% of muscle afferents traced from gastrocnemius and 40% of erector spinae. Sixty-five to 60% of TRPV1-positive muscles afferents also expressed CGRP. In contrast, expression of $P2X_3$ immnoreaction in TRPV1-positive muscle afferents were about 20%. TRPV1-positive primary afferents were contacted with spinal neurons expressing pERK after injection of acetic acid into the muscles. Conclusion: It is consequently suggested that nociception from skeletal muscles are mediated by TRPV1-positive primary afferents and majority of them are also peptidergic.

• 대한본초학회지
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• 제27권2호
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• pp.53-59
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• 2012

Objectives : This study was undertaken to investigate the effects of the GGEx18 ethyl acetate fraction (EF) on lipid accumulation and gene expression of fatty acid-oxidizing enzymes using 3T3-L1 adipocytes, C2C12 skeletal muscle cells, and NMu2Li liver cells. Methods : PPAR${\alpha}$, AMPK and UCPs transactivation was examined in NMu2Li hepatocytes, C2C12 myocytes, and 3T3-L1 preadipocytes using transient transfection assays. Results : 1. Compared with control, EF significantly increased the mRNA expression of VLCAD in 3T3-L1 adipocytes. 2. Compared with control, EF (0.1 ${\mu}g/ml$) significantly inhibited lipid accumulation in 3T3-L1 adipocytes. 3. EF significantly increased the mRNA expression of AMPK${\alpha}$1, AMPK${\alpha}$2 and PPAR${\alpha}$ in C2C12 skeletal muscle cells compared with control. 4. EF significantly increased the mRNA expression of genes involved in fatty acid ${\beta}$-oxidation, such as thiolase, MCAD, and CPT-1 in C2C12 skeletal muscle cells compared with control. 5. EF significantly increased the mRNA expression of UCP2 involved in energy expenditure in C2C12 skeletal muscle cells compared with control. 6. Compared with control, EF (10 ${\mu}g/ml$) significantly inhibited lipid accumulation in C2C12 skeletal muscle cells. 7. EF (10 ${\mu}g/ml$) significantly increased the mRNA expression of ACOX, HD, VLCAD and MCAD in NMu2Li liver cells compared with control. Conclusions : These results suggest that EF may prevent obesity by increasing the mRNA expression of mitochondrial fatty acid ${\beta}$-oxidizing enzymes in 3T3-L1 adipocytes, by not only regulating the fatty acid oxidation through activation of AMPK and PPAR${\alpha}$, but also increasing the UCP2 mRNA expression in C2C12 skeletal muscle cells, and by stimulating the mRNA expression of fatty acid-oxidizing enzymes in NMu2Li liver cells.

• 생명과학회지
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• 제13권3호
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• pp.324-332
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• 2003

최근 Phenobarbital에 의해서 발현되는 CYP2B유전자의 발현촉진 매개인자로서 Constitutive Androstane Receptor (CAR)가 최근에 규명되었다. 사람의 간암세포인 HepG2 cell line에 CAR유전자를 transfection시켰을 경우 PBRU의 활성을 촉진시켰다. 지금까지 연구된 CAR의 역할은 주로 간세포 내에서 cytochrome P4502B 유전자의 발현을 촉진하는 Phenobarbital의 매개체로서 알려져 있다. 다세포동물에서 각각의 분화된 세포는 독특한 유전자의 발현 Pattern을 갖고 있어서 어느 특정한 세포에서 일어나는 유전자의 발현특징이나 작용기전 혹은 기능이 다른 종류의 세포에서는 일어나지 않거나 상이한 기전에 의해서 조절된다. 이러한 세포간 특이성을 이용하여 유전자 발현에 관여하는 인자들을 규명하는데 기초적 자료로 이용될 수 있다. 따라서 본 연구의 목적은 CYP2B유전자의 발현이 일어나지 않는 콩팥세포 line (COS)에서 CAR 단백질 수용체가 PBRU의 활성촉진에 영향을 미치는지의 여부를 조사하고자 하였다. Control 상태의 Hep G2와 COS 배양세포에서는 CAR 단백질의 발현이 거의 나타나지 않았다. mCAR1-GFP를 transfection 한 후 CAR antibody를 이용하여 immunoblot을 시행하였을 경우, Hep G2 세포에서는 발현이 비교적 약하게 나타났지만, COS 세포에서는 강한 mCAR1-GFP 단백질의 발현을 보였다. 한편, GFP antibody를 이용하여 immunoblot을 시행하였을 경우에는 COS 세포에서 강한 mCAR1-GFP 단백질의 발현을 나타내었을 뿐만 아니라 Hep G2 세포에서도 명백히 단백질의 발현을 관찰할 수 있었다. 또한, Hep G2와 COS세포 모두에서 endogenous RXR의 발현이 일어남을 확인하였고 RXR expression plasmid를 transfection시켰을 때 두 세포 모두에서 단백질의 발현이 현저하게 증가되었다. Constitutive Androstane Receptor (CAR)에 의한 CYP2B의 PBRU 활성효과를 다르게 분화된 세포에서 차이가 일어나는지를 비교하기 위하여 CAR에 의해 매개되는 PBRU의 transactivation효과를 Hep G2와 COS세포에서 조사하였다. Hep G2 세포에서는 transfection된 CAR의 발현에 의해 firefly luciferase 보고단백질의 활성이 약 12배 증가하였다. CAR 발현유전자를 15 ng transfection하였을 때 주어진 보고유전자의 양에 대하여 최대반응을 나타내었고 CYP2B1PBRU가 제거된 CYP2C1 promotor/firefly luciferase를 보고유전자로 사용하였을 때는 CAR에 의한 luciferase의 활성이 나타나지 않았다. Hep G2와는 달리, COS세포에서는 transfection된 CAR의 발현이 PBRU에 의한 firefly luciferase보고단백질의 발현에 영향을 주지 못하였다. 이러한 결과들은 분화된 세포의 종류에 따라서 constitutive androstane receptor의 CYP2BPBRU 활성효과가 다르게 나타날 수 있음을 제시할 뿐만 아니라, 간세포에서 Phenobarbital에 의한 PBRU의 활성유도에 영향을 주는 endogenous 매개 인자들 중 CAR와 RXR과는 다른 전사조절인자들이 필요로 하며 이러한 인자들의 발현이 콩팥 세포에서는 다르게 존재함을 시사한다.