• Journal of Microbiology and Biotechnology
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• 제30권6호
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• pp.830-838
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• 2020

The histone-like nucleoid structuring protein (H-NS) is an abundant global regulator of environmentally controlled gene expression. Herein, we demonstrate that H-NS represses the expression of LeuO, the master regulator of the cyclic(Phe-Pro)-dependent signaling pathway, by directly binding to the upstream region of the gene. H-NS binds to a long stretched region (more than 160-bp long), which overlaps with binding sites for ToxR and LeuO. A high quantity of H-NS outcompetes ToxR for binding to the cis-acting element of leuO. However, our footprinting analyses suggests that the binding of H-NS is relatively weaker than LeuO or ToxR at the same molarity. Considering that the DNA nucleotide sequences of the upstream regions of leuO genes are highly conserved among various Vibrio, such patterns as those found in V. vulnificus would be a common feature in the regulation of leuO gene expression in Vibrionaceae. Taken together, these results suggest that, in species belonging to Vibrionaceae, H-NS regulates the expression of leuO as a basal stopper when cFP-ToxR mediated signaling is absent.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.59-67
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• 2015

동물의 장기를 인간에게 이식하게 되면 초급성거부반응(Hyperacute rejection, HAR)이 일어난다. 초급성거부반응은 면역계의 구성요소 중 보체(complement)에 의해 일어나는 거부반응으로 돼지의 혈관세포 표면에 있는 $Gal{\alpha}$(1,3)Gal 당분자에 인간의 항체가 즉각 반응하기 때문에 일어나며, ${\alpha}1,3$-galactosyltransferase(${\alpha}1,3$-GT) 유전자는 돼지 혈관세포 표면의 $Gal{\alpha}$(1,3)Gal 당분자 생성에 관여한다. 따라서 인간에게 돼지의 장기를 이식하기 위해서는 ${\alpha}1,3$-galactosyltransferase 유전자를 제거하는 것이 필요한 것으로 알려져 있다. 본 연구실의 이전 연구에서, 시카고 미니돼지 귀체세포에서 상동 재조합(Homologous recombination)을 통해 ${\alpha}1,3$-galactosyltransferase 유전자가 제거된 체세포를 개발한 바 있으며, 이 체세포를 통하여 ${\alpha}1,3$-GT 유전자가 제거된 돼지도 생산된 바 있다. 본 연구에서는, human serum 처리 시 돼지 세포를 보호해 준다고 보고되고 있는 human complement regulator인 human Decay-accelerating factor(hDAF)와 human ${\alpha}1,2$-fucosyltransferase(hHT)유전자를 ${\alpha}1,3$-GT 유전자 위치에 gene targeting하여 동시에 hDAF와 hHT가 발현하는 체세포를 개발하였다. Knock-in vector는 hDAF와 hHT 두 유전자가 발현할 수 있도록 IRES로 연결하였으며, ${\alpha}1,3$-GT 유전자의 start codon을 이용하여 발현할 수 있도록 구축하였다. 구축한 vector는 electroporation을 통해 미니 돼지 체세포에 도입하였으며, PCR 결과, ${\alpha}1,3$-GT 유전자 위치에서 상동 재조합이 일어났음을 확인하였다. Positive-negative 선별 방법을 통해 얻은 gene targeting 된 체세포는 RT-PCR에 의해 hDAF와 hHT 유전자의 발현이 확인되었으며, 대조군(NIH minipig)에 비해 ${\alpha}1,3$-GT 유전자의 발현이 감소하였다. 또한 이들 세포에 100% human complement serum을 처리하였을 때 knock-in 세포가 대조군에 비해 30% 정도 더 높은 생존율을 보였다. 따라서 개발된 체세포는 이종간 장기이식을 위한 돼지 생산과 함께 이를 이용한 이종간의 장기 이식 시 초급성 거부반응을 억제하는 데 사용될 수 있을 것으로 생각된다.

• 한국가축번식학회지
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• 제26권4호
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• pp.347-351
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• 2002

Transgenes in HSY-TK gene driven by the lck promoter was tested for the expression in immune cells (Jurkat cells) to apply xenotransplantation of human cells into transgenic animals for the potential use of the proliferation or differentiation of human stem cells in the large animal such as an pig. Also, lck-CFP gene was used for transfection experiment into Jurkat cell to confirm the proper regulation of lck promoter for transgene expression in the T cells. Transfection of lck-GFP gene into Jurkat ceils induced CFP expression in transfected cells. The expression of Ick-TK and Ick-CFP genes was confirmed by RT-PCR using RNAs extracted from Jurkat cells, When Jurkat cells transfected with TK and CFP genes were selected against G418 or gancyclovir treatments, Jurkat cells transfected with TK gene were not proliferated in G4i8 and gancyclovir medium while intact cells or cells transfected with CFP gene could grow in gancyclovir medium. However, Jurkat cells transfected with TK or GFP gene were proliferated in G418 medium probably due to Neo$^{r}$ gene in the vector. Gancyclovir treatment destroyed Jurkat cells expressing TK gene indicating that T-cells expressing TK gene can be selectively eliminated by TK gene expression driven by lck promoter.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.87-87
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• 2002

Previously, we observed high level expression of goat β-casein/genomic hGH fusion gene in mammary gland of transgenic mice. To develop an expression vector to make a human granulocyte-colony stimulating factor (hG-CSF) protein efficiently produced in milk of transgenic animals, we designed a new bicistronic vector using the goat β-casein/genomic hGH fusion gene as regulation sequences for expression and internal ribosome entry site (IRES) as a mediator for second gene expression. This vector was constructed by insertion of encephalomyocarditis virus (EMCV) IRES-dependent second gene region coupled with hG-CSF cDNA into 3' untranslated region of an intact hGH gene. By microinjcetion, four transgenic mice were generated and three of them transmitted the bicistronic vector to their progeny. (omitted)

• Journal of Veterinary Science
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• 제23권2호
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• pp.24.1-24.10
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• 2022

Background: Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. Objectives: The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. Methods: The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. Results: The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. Conclusions: These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.

• 동의생리병리학회지
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• 제18권5호
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• pp.1291-1300
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• 2004

Baicalin, a biologically active flavonoid form the roots of Scutallaria baicalensis (Skullcap), have been reported to not only function as anti-oxidants but also cause anticancer effect. We investigated the mechanism of baicalin-induced cytotoxicity and the macro scale gene expression analysis in leukemia cell line, HL-60 cells. Baicalin (10 μM) were used to treat the cells for 6h, 12h, 24h, 48h and 72h. In a human cDNAchip study of 65,000 genes evaluated 6, 12, 24, 48. 72 hours after treated with Baicalin in HL-60 cells. Hierarchical cluster against the genes which showed expression changes by more than two fold. One hundred one genes were grouped into 6 clusters according to their profile of expression by a hierarchical clustering algorithm. For genes differentially expressed in response to baicalin treatment, we tested functional classes based on Gene Ontology (GO) terms. This study provides the most comprehensive available survey of gene expression changes in response to baicalin treatment in HL-60 cell line.

• Natural Product Sciences
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• 제21권1호
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• pp.59-65
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• 2015

In this study, we investigated whether cynaroside, cynarin and linarin derived from Chrysanthemum indicum L. affect the secretion, production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with cynaroside, cynarin or linarin for 30 min and then stimulated with PMA (phorbol 12-myristate 13-acetate) for 24 h. The MUC5AC mucin gene expression, mucin protein production and secretion were measured by RT-PCR and ELISA, respectively. Effect of linarin on EGF (epidermal growth factor) - or TNF-${\alpha}$ (tumor necrosis factor-${\alpha}$)-induced MUC5AC mucin gene expression and mucin protein production was also examined. The results were as follows: (1) Cynaroside and cynarin did not significantly affect PMA-induced MUC5AC mucin secretion from NCI-H292 cells. However, linarin decreased MUC5AC mucin secretion; (2) Cynaroside did not affect PMA-induced MUC5AC mucin production and gene expresion from NCI-H292 cells. However, cynarin and linarin inhibited the production and gene expression of MUC5AC mucin; (3) Linarin also inhibited the production and gene expression of MUC5AC mucin induced by EGF- or TNF-${\alpha}$ from NCI-H292 cells. These results suggest that linarin can regulate the gene expression, production and secretion of mucin, by directly acting on airway epithelial cells.

• Asian-Australasian Journal of Animal Sciences
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• 제19권8호
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• pp.1085-1089
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• 2006

The objectives of this study were to confirm a location of the calpastatin (CAST) gene in chromosome 2 and to detect associations of genetic variations with economic traits in the porcine CAST gene as a candidate gene for growth and meat quality traits in pigs. Calpastatin is a specific endogenous inhibitor of calpains. The calpain protease system is ubiquitous, and is involved in numerous growth and metabolic processes. Three single nucleotide variations were identified within a 1.6 kb fragment of the porcine CAST gene and these polymorphisms were used for genetic linkage mapping. Linkage and QTL mapping were performed with the National Livestock Research Institute (NLRI) reference families using eight microsatellites and SNP makers in the CAST gene. The porcine CAST gene was mapped adjacent to the markers, SW395 and SW1695 on SSC2 with LOD scores of 15.32 and 8.50, respectively. According to the QTL mapping, a significant association was detected at 82 cM between SW395 and CAST-Hinf I for weight at the age of 30 weeks. In addition, an association study was performed with the $F_2$ animals of NLRI reference families for Hinf I, Msp I and Rsa I polymorphisms in the CAST gene. Two polymorphisms, CAST-Rsa I and CAST-Hinf I, showed significant correlation for growth traits at p<0.01 and p<0.05, respectively.

• 한국가축번식학회지
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• 제27권3호
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• pp.197-206
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• 2003

hFSH는 $\alpha$와 $\beta$ subunit으로 구성된 heterodimer로서 두 subunit의 조합은 활성을 지닌 호르몬의 생산에 있어서 매우 중요한 단계이다. 이 조합과정의 효율을 증대하기 위하여 본 연구에서는 hFSH를 단일사슬의 단백질로 구축하고자 하였으며, 이의 일환으로 각 subunit 대한 cDNA단편을 연결하는 서열로 CTP linker를 도입하였다. 재조합한 hFSH-CTP 유전자는 pseudotype의 retrovirus vector system을 이용하여 CHO 세포와 닭의 배로 각각 전이되었다. CHO 세포에서의 FSH의 생산은 $\alpha$와 $\beta$를 각각 전이한 경우에 비해 hFSH-CTP를 전이한 경우에서 17배 이상 높은 것으로 나타났다. 닭에서는 유전자 전이를 시도한 62개체 중에서 11마리가 부화하였으며 그 중 10마리가 형질전환된 닭인 것으로 RT-PCR을 통하여 확인되었다. 그러나 개체의 혈중 FSH의 생산은 확인하지 못하였다. 이상의 실험 결과를 바탕으로 하여 재조합된 hFSH-CTP는 FSH의 발현에 매우 효율적인 구조로 생각되며, 또한 retrovirus를 이용한 유전자 전이 방법은 형질전환 가금의 생산에 있어서 매우 적절한 방법으로 사료된다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.120-120
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• 1995

The total size of the pF AR1, a genomic clone of Frankia FaCI, was estimated to be about 44Kb by summation of the individual fragment length generated by single or double restriction enzymes. Southern hybridization analyses with Azotobacter vinelandii nif-genes as probes and partial sequencing analyses of the subclones revealed that organization of the nif-gene in the FaCI strain was nifV, H, D, K, E, N, X, W, B. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes but the positioning of the nifV-like gene relative to the nifHDK cluster differs. A consensus nif-promoter-like sequence, found at 5' of nifH, was not detected upstream of the niJV-like gene. nifV-like gene contained a ORF of 1206 NT encoding 401 amino acids. The nucleotide sequence and deduced amino acid sequence of the gene exhibit homology value of 65% and 41% with that from A vinelandii, respectively. The putative Shine-Dargamo sequences were present preceding nitK, nifH, D, K, and nifE, and in nitK gene putative start codon GTG was detected instead of A TG. The nucleotide and amino acid sequence of niIK of FaCI showed 82% and 76% homolgy with those of Frankia HFPCc 13, respectively. Amino acid sequence of niIK showed 69% and 61% homology with those of A vinelandii, Klebsiella pnewnoniae, respectively, while that of nifE 73% and 71%, respecti vely.i vely.