• 제목/요약/키워드: Exogenous Genes

검색결과 147건 처리시간 0.029초

Inducible Expression of the Lactadherin Gene with a Reverse Tetracycline-Regulated Retroviral Vector System (Tetracycline으로 발현이 유도되는 Retrovirus Vector System을 이용한 Human Lactadherin 유전자의 전이와 발현)

  • 이용석;오훈규;권모선;박창식;김태완;박재복
    • Korean Journal of Animal Reproduction
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    • 제27권3호
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    • pp.259-268
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    • 2003
  • Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinoma cells and is known to prevent symptomatic rotavirus infections. In this study, we tried to transfer the human lactadherin gene to the Chinese Hamster Ovary (CHO) cells using retrovirus vector system and tested inducible expression of the gene under the tetracycline-controllable promoter. At first, tetracycline-mediated inducibility was tested using E.coli LacZ marker gene. NIH3T3 cells co-infected with RevTet-On and RevTRE-LacZ retrovirus vectors showed that the cells responded to doxycycline (a derivative of tetracycline) in a dose-dependent manner, and prominent induction of the lacZ gene expression was observed from 1 $\mu\textrm{g}$/ml of doxycycline concentration. Based on the results of the pilot experiment, inductional expression of the human lactadherin gene was conducted using RevTet-On and RevTRE-Ltd retrovirus vectors. Analysis with the RT-PCR demonstrated successful inductional expression of the lactadherin gene in the Chinese Hamster Ovary (CHO) cells. Considering that constitutive overexpression of the exogenous genes in the target cells causes serious physiological imbalance, the results obtained in this study will be very useful especially in the studies of gene therapy and transgenic animal production.

Metabolic Engineering of the Brassinosteroid Biosynthetic Pathways (Brassinosteroid의 대사공학)

  • LEE Mi-Ock;SONG Ki-Hong;LEE Hyun-Kyung;JUNG Ji-Yoon;CHOE Vit-Nary;CHOE Sunghw
    • Proceedings of the Korean Society of Plant Biotechnology Conference
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    • 한국식물생명공학회 2002년도 추계학술대회
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    • pp.69-75
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    • 2002
  • Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd/dwf3 were shown to be blocked in $D^4$ reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bri1/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus It is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRI1 could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

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A Study of the Generation of Transgenic Chickens That Express Human SOD-3 Protein (사람의 SOD-3 단백질을 발현하는 형질전환 닭 생산 연구)

  • Byun, S.J.;Park, C.;Kim, J.A.;Woo, J.S.;Lee, H.C.;Kim, T.Y.;Kim, S.H.;Seong, H.H.;Park, J.K.;Jeon, I.S.
    • Korean Journal of Poultry Science
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    • 제35권3호
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    • pp.241-245
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    • 2008
  • Lentiviral vector system is efficient vehicles for the delivery of exogenous genes, and it is generally used in the generation of transgenic chickens. In this study, we used recombinant lentiviral vectors to generate transgenic chicks that express the human superoxide dismutase-3 gene driven by the chicken ovalbumin promoter. It is well known that superoxide dismutases(SODs) are believed to play a crucial role in protecting cells against oxygen toxicity. There are three forms of SOD proteins: cytosolic Cu-Zn SOD, mitochondrial Mn SOD, and extracellular SOD(SOD-3). The recombinant lentivirus containing the human SOD-3 gene was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. From 341 injected embryos, the 78 chicks hatched after 21 days incubation. The hatched chicks were screened for the human SOD-3 gene by using PCR. Two of 47 male chickens that survived to sexual maturity contained the human SOD-3 gene in their semen. These results showed that our transgenic chicken generation system was completely established.

Compound K improves skin barrier function by increasing SPINK5 expression

  • Park, No-June;Bong, Sim-Kyu;Lee, Sullim;Jung, Yujung;Jegal, Hyun;Kim, Jinchul;Kim, Si-Kwan;Kim, Yong Kee;Kim, Su-Nam
    • Journal of Ginseng Research
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    • 제44권6호
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    • pp.799-807
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    • 2020
  • Background: The skin acts as a barrier to protect organisms against harmful exogenous agents. Compound K (CK) is an active metabolite of ginsenoside Rb1, Rb2 and Rc, and researchers have focused on its skin protective efficacy. In this study, we hypothesized that increased expression of the serine protease inhibitor Kazal type-5 (SPINK5) may improve skin barrier function. Methods: We screened several ginsenosides to increase SPINK5 gene promoter activity using a transactivation assay and found that CK can increase SPINK5 expression. To investigate the protective effect of CK on the skin barrier, RT-PCR and Western blotting were performed to investigate the expression levels of SPINK5, kallikrein 5 (KLK5), KLK7 and PAR2 in UVB-irradiated HaCaT cells. Measurement of transepidermal water loss (TEWL) and histological changes associated with the skin barrier were performed in a UVB-irradiated mouse model and a 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis-like model. Results: CK treatment increased the expression of SPINK5 and decreased the expression of its downstream genes, such as KLKs and PAR2. In the UVB-irradiated mouse model and the DNCB-induced atopic dermatitis model, CK restored increased TEWL and decreased hydration and epidermal hyperplasia. In addition, CK normalized the reduced SPINK5 expression caused by UVB or DNCB, thereby restoring the expression of the proteins involved in desquamation to a level similar to normal. Conclusions: Our data showed that CK contributes to improving skin-barrier function in UVB-irradiated and DNCB-induced atopic dermatitis-like models through SPINK5. These results suggest that therapeutic attempts with CK might be useful in treating barrier-disrupted diseases.

Effects of Abscisic Acid (ABA) and Fluridone on Red Coloration of 'Hongro' Apple Fruit Skins (Abscisic acid(ABA) 및 fluridone의 처리가 'Hongro' 사과의 과피 착색에 미치는 영향)

  • Ryu, Suhyun;Kwon, Yong Hee;Do, Gyeong Ran;Jeong, Jae Hoon;Han, Hyun Hee;Han, Jeom Hwa
    • Journal of Bio-Environment Control
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    • 제25권4호
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    • pp.240-248
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    • 2016
  • The objective of this study was to determine the effect of exogenous abscisic acid (ABA) on the red coloration and endogenous ABA contents of apple fruit skins. ABA and fluridone (an ABA synthetic inhibitor, FD) was sprayed on 'Hongro' apple fruit skins at 107 days after full bloom (DAFB). Visual coloration and hunter's color values were not affected by the ABA and FD treatments. Anthocyanin contents in fruit skins increased similarly to hunter $a^*$ values of fruit skins, but ABA and FD did not affect its accumulations. Liquid chromatography analysis revealed that endogenous ABA contents in control fruit increased at first and then decreased from 12 hours after the treatment. ABA treatment increased ABA contents in fruit skins from 2 hour after the treatment and it lasted until the end of the treatments. FD decreased ABA contents in fruit skins from 6 hours after the treatment. ABA treatment increased MdNCED2 (an ABA biosynthetic gene), MdACO1 (an ethylene biosynthetic gene), and MdCHS and MdDFR expressions. However, MdUFGT expressions were not affected by ABA treatment.

Metabolic Engineering of the Brassinosteroid Biosynthetic Pathways (Brassinosteroid의 대사공학)

  • Lee, Mi-Ock;Song, Ki-Hong;Lee, Hyun-Kyung;Jung, Ji-Yoon;Choe, Vit-Nary;Choe, Sung-Hwa
    • Proceedings of the Korean Society of Plant Biotechnology Conference
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    • 한국식물생명공학회 2002년도 춘계학술대회
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    • pp.69-75
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    • 2002
  • Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd/dwf3 were Shown to be blocked in $D^4$ reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bri1/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRI1 could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

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Roles of CYP1A1 and CYP2E1 Gene Polymorphisms in Oral Submucous Fibrosis

  • Yaming, Punyo;Urs, Aadithya Basavaraj;Saxena, Alpana;Zuberi, Mariyam
    • Asian Pacific Journal of Cancer Prevention
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    • 제17권7호
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    • pp.3335-3340
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    • 2016
  • Background: Oral submucous fibrosis (OSF) is a precancerous condition with a 4 to13% malignant transformation rate. Related to the habit of areca nut chewing it is mainly prevalent in South-east Asian countries where the habit of betel quid chewing is frequently practised. On chewing, alkaloids and polyphenols are released which undergo nitrosation and give rise to N-nitrosamines which are cytotoxic agents. CYP450 is a microsomal enzyme group which metabolizes various endogenous and exogenous chemicals including those released by areca nut chewing. CYP1A1 plays a central role in metabolic activation of these xenobiotics, whereas CYP2E1 metabolizes nitrosamines and tannins. Polymorphisms in genes that code for these enzymes may alter their expression or function and may therefore affect an individuals susceptibility regarding OSF and oral cancer. The present study was therefore undertaken to investigate the association of polymorphisms in CYP1A1 m2 and CYP2E1 (RsaI/PstI) sites with risk of OSF among areca nut chewers in the Northern India population. A total of 95 histopathologically confirmed cases of OSF with history of areca nut chewing not less than 1 year and 80, age and sex matched controls without any clinical signs and symptoms of OSF with areca nut chewing habit not less than 1 year were enrolled. DNA was extracted from peripheral blood samples and polymorphisms were analyzed by PCR-RFLP method. Gene polymorphism of CYP1A1 at NcoI site was observed to be significantly higher (p = 0.016) in cases of OSF when compared to controls. Association of CYP1A1 gene polymorphism at NcoI site and the risk of OSF (Odd's Ratio = 2.275) was also observed to be significant. However, no such association was observed for the CYP2E1 gene polymorphism (Odd's Ratio = 0.815). Our results suggest that the CYP1A1 gene polymorphism at the NcoI site confers an increased risk for OSF.

Metabolic Engineering of the Brassinosteroid Biosynthetic Pathways (Brassinosteroid의 대사공학)

  • Lee, Mi-Ock;Song, Ki-Hong;Lee, Hyun-Kyung;Jung, Ji-Yoon;Choe, Vit-Nary;Choe, Sung-Hwa
    • Journal of Plant Biotechnology
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    • 제29권2호
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    • pp.139-144
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    • 2002
  • Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd /dwf3 were shown to be blocked in D$^4$reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bril/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRIl could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

Investigation of ginsenosides in different tissues after elicitor treatment in Panax ginseng

  • Oh, Ji Yeon;Kim, Yu-Jin;Jang, Moon-Gi;Joo, Sung Chul;Kwon, Woo-Saeng;Kim, Se-Yeong;Jung, Seok-Kyu;Yang, Deok-Chun
    • Journal of Ginseng Research
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    • 제38권4호
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    • pp.270-277
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    • 2014
  • Background: The effect of methyl jasmonate (MJ) on ginsenoside production in different organs of ginseng (Panax ginseng Meyer) was evaluated after the whole plant was dipped in an MJ-containing solution. MJ can induce the production of antioxidant defense genes and secondary metabolites in plants. In ginseng, MJ treatment in adventitious root resulted in the increase of dammarenediol synthase expression but a decrease of cycloartenol synthase expression, thereby enhancing ginsenoside biosynthesis. Although a previous study focused on the application of MJ to affect ginsenoside production in adventitious roots, we conducted our research on entire plants by evaluating the effect of exogenous MJ on ginsenoside production with the aim of obtaining new approaches to study ginsenoside biosynthesis response to MJ in vivo. Methods: Different parts of MJ-treated ginseng plants were analyzed for ginsenoside contents (fine root, root body, epidermis, rhizome, stem, and leaf) by high-performance liquid chromatography. Results: The total ginsenoside content of the ginseng root significantly increased after 2 d of MJ treatment compared with the control not subjected to MJ. Our results revealed that MJ treatment enhances ginsenoside production not in the epidermis but in the stele of the ginseng root, implying transportation of ginsenosides from the root vasculature to the epidermis. Application of MJ enhanced protopanaxadiol (PPD)-type ginsenosides, whereas chilling treatment induced protopanaxatriol (PPT)-type ginsenosides. Conclusion: These findings indicate that the production of PPD-type and PPT-type ginsenosides is differently affected by abiotic and biotic stresses in the ginseng plant, and they might play different defense mechanism roles.

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THE GENE FOR THE HEMIN-BINDING PROTEIN FROM Prevotella intermedia (Prevotella intermedia에서의 Hemin 결합 단백질 유전자의 분리 및 염기서열 분석)

  • Kim, Shin;Kim, Sung-Jo
    • Journal of the korean academy of Pediatric Dentistry
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    • 제33권2호
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    • pp.304-310
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    • 2006
  • Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$, and screened for recombinant clones using heminbinding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phonotype. The 2.5kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.

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