• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.544-550
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• 2005

To develop a highly efficient mammalian expression vector, a series of vectors were constructed based on the murine cytomegalovirus (MCMV) immediate-early (IE) promoter and human ${\beta}$-globin second intron. The resulting MCMV promoter was several-fold stronger than the HCMV promoter in various mammalian cell lines, such as the NIH3T3, Neuro-2a, 293T, and HT1080 cell lines, and was only slightly weaker than the HCMV promoter in HeLa and CHO cells. The inclusion of the human ${\beta}$-globin second intron behind the MCMV promoter or HCMV promoter markedly enhanced the promoter activity in various mammalian cell lines, and the resultant MCMV/Glo-I expression system was stronger than the HCMV promoter from 4.7- to 11.2-fold in every cell line tested. Also, the MCMV/Glo-I promoter induced a higher level of the VSV-G protein in a transiently transfected 293T cell line, which is useful for the production of recombinant retrovirus and lentivirus vectors.

• BMB Reports
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• 제29권4호
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• pp.359-364
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• 1996

By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.

• KSBB Journal
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• 제5권1호
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• pp.49-58
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• 1990

대장균으로부터 alpha interferon의 생산과 분비를 유도하기 위해 대장균의 lipoprotein promoter, lactose promoter 및 operator와 lipoprotein의 signal seqquence를 가지는 vector에 alpha-IFN유전자를 cloning하여 발현 vector pIF-III-B와 vector pIF-III-C를 제작하였다.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.260-267
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• 2008

단백질의 산업적 생산을 위해 발현벡터의 선정이 중요하지만 이용 가능한 프로모터가 극히 제한적이며 많은 경우 과발현되는 특성과 함께 불용성 응집체가 형성되는 단점을 지닌다. 따라서 다양한 생물로부터 유래된 잠재성이 큰 유전자원(metagenome)에서의 프로모터 발굴과 한정된 숙주를 해결하려는 노력이 요구된다. 선행연구에서 발굴한 metagenome 유래의 항시발현 프로모터를 이용해 대장균의 일반적인 배양조건에서 세포생리에 영향이 적은 신규 항시발현 벡터를 제작하였다. 이를 위해 예측된 프로모터 서열과 MCS를 포함하는 합성 primer를 제작한 후 PCR로 증폭해 발현벡터를 구성한 후, 프로모터 구동여부와 단백질 발현양상 등을 관찰하였다. 인위적으로 도입된 MCS에 GFP, esterase, $\beta$-glucosidase를 클로닝해 단백질 발현양과 가용성을 분석한 결과, 안정적으로 전체단백질의 $2{\sim}3%$ 정도로 발현되며 80% 이상의 높은 가용성을 지닌 단백질의 발현이 유도되는 것으로 확인되었다. 이와 같은 결과는 잠재적인 생물자원의 보고로서 metagenome의 활용가능성을 제시하고 있다. 따라서 다양한 숙주에서 작동하는 프로모터의 발굴 및 발현벡터의 제작을 시도할 경우 단백질의 생산이나 대사공학에 의한 균주개량에 유용하게 활용할 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• 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.

• 생명과학회지
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• 제19권6호
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• pp.809-817
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• 2009

식물에서 유전자의 기능을 연구하는데 있어서 유전자가 과발현 되거나 발현이 억제되는 형질전환체는 해당 유전자의 기능과 관련되어 매우 유용한 정보를 제공한다. 본 연구에서는 modified CaMV 355, UBQ3, UBQ10 프로모터를 pPZP211 벡터에 각각 클로닝 하여 Agrobacterium을 매개로 한 과발현형질전환 식물체 제작에 유용하게 이용할 수 있는 pFGL571, pFGL846, pFGL847을 제조하였다. 이 벡터들은 크기가 작고, 박테리아 내에 high copy로 존재하며, 다중 클로닝 부위에 다양한 제한효소 부위를 가지고 있고, 전체 서열이 알려져 있는 등의 장점을 가지고 있다. GUS 또는 sGFP 리포터 유전자를 포함하는 형질전환 식물체를 제조하여 modified CaMV 35S, UBQ3, UBQ10 프로모터의 활성을 분석한 결과, 세 프로모터 모두 발아 후 대부분의 발달단계와 성숙한 식물체의 꽃 기관에서 높은 활성을 보였다. 한편, 식물에서 유전자 발현 억제에 이용할 수 있는 RNAi 기본 벡터인 pFGL727을 제조하였고, pFGL727을 이용한 벼 RNAi 형질전환체의 분석을 통해 이벡터가 유전자의 발현 억제에 유용하게 이용될 수 있음을 확인하였다. 연구 결과를 종합해 보면, 본 연구에서 제조한 벡터들은 식물에서 유전자 과발현과 발현 억제에 유용하게 이용될수 있을 것으로 기대된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.162-165
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• 2003

In order to study the secretion of the human urokinase-type plasminogen activator, u-PA, from the yeast yarrowia lipolytica, three kinds of integrative expression vector were constructed. These vectors differed only in their secretion control legions, pre-, pre-dip-(dipeptide Stretch) or pre-dip-pro sequences of the alkaline extracellular protease, which were joined inflame to the human u-PA cDNA. The recombinant Y. lipolytica Strains, transformed with the expression vectors, secreted the hyperglycosylated u-PA. A fibrin plate assay of the culture supernatants showed that the hyperglycosylated u-PA proteins could catalyze fibrinolysis, and that the pre-dip sequence was the most efficient secretory signal for the secretion of the u-PA from Y. lipolyica. This result suggests that Y. lipolytica can be developed as a potential host for the production of recombinant human u-PA.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.23-28
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• 2005

The high-dimensionality and insufficiency of gene expression profiles and proteomic profiles makes feature selection become a critical step in efficiently building accurate models for cancer problems based on such data sets. In this paper, we use a method, called Discrete Function Learning algorithm, to find discriminatory feature vectors based on information theory. The target feature vectors contain all or most information (in terms of entropy) of the class attribute. Two data sets are selected to validate our approach, one leukemia subtype gene expression data set and one ovarian cancer proteomic data set. The experimental results show that the our method generalizes well when applied to these insufficient and high-dimensional data sets. Furthermore, the obtained classifiers are highly understandable and accurate.

• 제어로봇시스템학회논문지
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• 제13권8호
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• pp.754-759
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• 2007

In this paper, we proposed the Bi-Modal Sensor Fusion Algorithm which is the emotional recognition method that be able to classify 4 emotions (Happy, Sad, Angry, Surprise) by using facial image and speech signal together. We extract the feature vectors from speech signal using acoustic feature without language feature and classify emotional pattern using Neural-Network. We also make the feature selection of mouth, eyes and eyebrows from facial image. and extracted feature vectors that apply to Principal Component Analysis(PCA) remakes low dimension feature vector. So we proposed method to fused into result value of emotion recognition by using facial image and speech.