• 농약과학회지
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• 제2권1호
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• pp.91-96
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• 1998

모기에 독성을 보이는 cry11Aa 유전자를 발현시키기 위해 cyanobacteria와 E. coli에서 발현 될 수 있는 두 가지 상이한 벡터 (pCYASK5-1, pCYASK5-2)를 제작하였다. 구축한 두 벡터를 E. coli에 형질전환하여 cry11Aa 유전자의 발현을 SDS-polyacrylamide gel electrophoresis (PAGE)와 Western blot analysis을 통해 조사한 결과, pCYASK5-1와 pCYASK5-2으로 형질전환된 E. coli는 각각 72kDa과 64kDa 크기의 cry11Aa 유전자를 발현하였다. 형질전환체의 모기살충성을 조사한 결과, pCYASK5-1과 pCYASK5-2을 가지는 형질전환체는 빨간집 모기유충(Culex pipiens)에 대해 각각 93%, 89%의 치사율을 보였다.

• 한국작물학회지
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• 제48권1호
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• pp.20-24
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• 2003

Proline is known as an osmotrotectant to enhance tolerance against both salt and dehydration stresses. A P5CS ($\Delta^1$-pyrroline-5-carboxylate synthetase) plays a major role in regulation of synthesis of proline. An overexpression of the mothbean P5CS gene in transgenic tobacco plant increased the levels of proline and osmotolerance. In an attempt to look for the possibility to use content of proline as well as a level of P5CS gene expression as molecular markers for salt tolerance, the amounts of proline and transcript levels of P5CS were measured as functions of either concentration of NaCl or length of treatment period among different species of zoysiagrass. Hybridzoysia showed the highest level of proline ($329\mu\textrm{g}$/g.f.w.) among five different species of zoysiagrass at 250 mM NaCl in 24 hours. The level of P5CS transcript was also the highest in the hybridzoysia at 250 mM NaCl in 24 hours. The transcriptions of P5CS gene were induced at the rates of 1.2, 1.2, 1.8, and 1.8, upon treatment of 250 mM NaCl in Z. japonica, Z. matrella, Z. sinica and hybridzoysia respectively. Based on a correlation between the level of P5CS transcript and the proline content among different species of zoysiagrass, a comparative structural analysis of the gene for P5CS from either Z. sinica or hybridzoysia may lead to an understanding of mechanism for salt tolerance shown differently among zoysiagrasses.

• 대한의생명과학회지
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• 제26권2호
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• pp.114-119
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• 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.

• 농업과학연구
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• 제48권2호
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• pp.353-365
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• 2021

Previously, we reported that tissue factor (Tf) was included in the list of differentially expressed genes as an upregulated gene in a rejected porcine heart after xenotransplantation into monkey. In this study, we analyzed that expression of Tf in aortic endothelial cells (pAEC) isolated from alpha 1,3-galactosyltransferase knockout pig in response to allogeneic porcine serum and xenogeneic human serum. The consequence was significant upregulation of Tf expression by responding to human serum compared with porcine serum. To analyze the function of Tf gene as a promoter, we constructed reporter vectors for expression of luciferase linked to 1,246 and 787 base pairs of porcine Tf (pTF1246 and pTF787), and 535 base pairs of human TF (hTF535) sequences including putative promoter regions and AP-1 biding site at the 5' end. The reporter vectors were transfected into pAEC including cytomegalovirus enhancer/chicken β-actin (CAG)-luciferase vector as a control. Luciferase assay showed that all of the promoters were insufficient to express luciferase compared with CAG promoter in basic culture conditions. Notably, pTF1246, pTF787, and hTF535 led to a significant increase of luciferase expression in response to human serum compared with porcine serum while no change of CAG. pTF1246 and pTF787 showed higher expression than hTF535. Taken together, our findings suggest that pTF1246 and pTF787 promoters could mediate target gene expression specifically at xenogeneic stress condition.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.9-14
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• 1999

Factors affecting gene gun-mediated expression of the human erythropoietin gene were investigated in primary cultured oviduct cells from laying hens. The human erythropoietin gene was transfected by a gene gun method at $1.25{\mu}g$ per dish, and cultured in a synthetic serum-free medium for 72 hrs. The concentration of human erythropoietin mRNA was determined by RNA : RNA solution hybridization. In experiment 1, the effect of changing the shooting pressure of DNA-coated microparticles with nitrogen gas was tested at 20 and $60kgf/cm^2$. The results showed that the erythropoietin mRNA concentration was significantly higher at 60 than $20kgf/cm^2$. In experiment 2, the effects of supplementing the medium with fetal calf serum at 10%, and raising the shooting pressure from 60 to $80kgf/cm^2$ on the cell number and erythropoietin gene expression were examined. Although supplementation with fetal calf serum significantly increased the cell numbes compared with no supplemented controls (p < 0.05), erythropoietin mRNA concentration per $10^3$ cells was not affected. Raising the shooting pressure from 60 to $80kgf/cm^2$ did not affect either of the parameters, In experiment 3, the effect of supplementing ascorbate 2-phosphate at 0.5 mM was tested. The results indicated that the ascorbate supplementation significantly increased the cell number (p < 0.05), and tended to increase erythropoietin mRNA concentration (p < 0.1). Thus, for human erythropoietin gene expression by using the gene gun method, shooting pressure with nitrogen gas should be sufficient at $60kgf/cm^2$ and supplementation with ascorbate phosphate would be useful to enhance not only the cell proliferation but also erythropoietin gene expression.

• 한국미생물·생명공학회지
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• 제14권2호
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• pp.155-160
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• 1986

E. coli-Yeast shuttle vector YEp 13에 B. amyloliquefaciens의 $\alpha$-amylase gene을 cloning하여 얻은 hybrid plasnidlasmid를 E. coli를 숙주세포로 하여 형질을 발현시켰다. 형질전환주의 $\alpha$-amylase 활성 측정 결과, B. amyloliquefaciens에 대해 20-30%의 상대 활성도를 나타내었다. 형질전환주의 경우 생성된 $\alpha$-amylase의 60-65%가 periplasm에 축적되었으며 세포 외부로의 분비는 없었다. Hybrid DNA를 agarose-gel 전기영동으로 조사한 결과 그 크기가 다른 다수의 hybrid DNA가 확인되었으며 pHA28의 plasmid (a)(Fig.3)에서만 $\alpha$-amylase 활성을 나타내는 것으로 밝혀졌다 pHA28의 plasmid(a)의 크기가 YEp 13 plasmid DNA보다 작은 것은 YEp13 plasmid에서 yeast gene부분이 deletion된 결과로 추측되었다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5241-5243
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• 2012

Background: Prostate cancer (Pca) is one of the most common complex and polygenic diseases in men. The X-ray repair complementing group 1 gene (XRCC1) is an important candidate in the pathogenesis of Pca. The purpose of this study was to evaluate the association between single nucleotide polymorphisms in the XRCC1 gene and susceptibility to Pca. Materials and Methods: XRCC1 gene polymorphisms and associations with susceptibility to Pca were investigated in 193 prostate patients and 188 cancer-free Chinese men. Results: The c.910A>G variant in the exon9 of XRCC1 gene could be detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods. Significantly increased susceptibility to prostate cancer was noted in the homozygote comparison (GG versus AA: OR=2.95, 95% CI 1.46-5.42, ${\chi}^2$=12.36, P=0.001), heterozygote comparison (AG versus AA: OR=1.76, 95% CI 1.12-2.51, ${\chi}^2$=4.04, P=0.045), dominant model (GG/AG versus AA: OR=1.93, 95% CI 1.19-2.97, ${\chi}^2$=9.12, P=0.003), recessive model (GG versus AG+AA: OR=2.17, 95% CI 1.33-4.06, ${\chi}^2$=8.86, P=0.003) and with allele contrast (G versus A: OR=1.89, 95% CI 1.56-2.42, ${\chi}^2$=14.67, P<0.000). Conclusions: These findings suggest that the c.910A>G polymorphism of the XRCC1 gene is associated with susceptibility to Pca in Chinese men, the G-allele conferring higher risk.

• BMB Reports
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• 제39권1호
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• pp.22-25
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• 2006

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

• 대한수의학회지
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• 제32권4호
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• pp.555-567
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• 1992

In this study gene encoding structural proteins of a CPV isolate was cloned and sequenced to elucidate the molecular genetical properties of the canine parvoviruses isolated from the field. Six recombinant plasmids of pEP3, p1471, p2070, pEP069, pEP338 and p14711p were constructed from the map positions 22 to 98 of RF DNA to clone the VP1 and VP2 genes of CPV-V20. Sequentialy the gene comprising 3780 nucleotides were sequenced by dideoxy chain termination method. When nucleotide sequence of gene encoding the structural proteins of CPV-V20 was compared with those of other strains, CPV-N, CPV-d and CPV-780929 published previously, DNA, homologies to CPV-V20 were 99.87% with CPV-N, 99.73% with CPV-d, 96.85% with CPV-780929 and 98.4% with FPLV-Carl, respectively. The DNA sequence data of CPV-V20 showed seven point mutations and also deletion of 135 nucleotides from the nucleotide position 4745 to 4879 located in the 3'-noncoding region of CPV-N.

• Journal of Plant Biology
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• 제39권4호
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• pp.243-247
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• 1996

Phosphoribosylanthranilate transferase (PAT) catalyzes the second step of the tryptophan biosynthetic pathway and is encoded by a single-copy gene that complements all the visible phenotypes of the tryptophan mutant (trp1-100) of Arabidopsis. The trp1-100 is blue fluorescent under UV light becuase it accumulates anthranilate. To obtain a plant with reduced PAT activity, PAT1 genes with several internal deletions in different promoter regions (pHS 101, pHS102, pHS104, pHS105, and pHS107) were induced into trp1-100 via Agrobacterium. Then, homozygous T3 plants were isolated and examined for blue fluorescence. Introduction of the PAT1 gene fusants results in the reversion of fluorescence phenotype except in the case of pHS105. These results prompted us to perform a parallel analysis of anthranilate synthase and PAT interms of the genetic complementation. A plant line carrying pHS105 gene fusant does not completely complement the blue fluorescence but it accumulates less anthranilate than trp1-100. The activity of PAT was reduced in the transgenic mutant as well. The plant carrying these constructs will add to the growing collection of molecular tools for the study of the indolic secondary metabolism.