• 대한의생명과학회지
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• 제13권3호
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• pp.239-244
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• 2007

Hoxc8 is one of the homeotic developmental control genes regulating the expression of many downstream target genes, through which animal body pattern is established during embryonic development. In previous proteomics analysis, proliferating cell nuclear antigen (PCNA) which is also known as cyclin, has been implied to be regulated by Hoxc8 in F9 murine embryonic teratocarcinoma cell. When the 5' upstream region of PCNA was analyzed, it turned out to contain 20 Hox core binding sites (ATTA) in about 1.17 kbp (kilo base pairs) region ($-520{\sim}-1690$). In order to test whether this region is responsible for Hoxc8 regulation, the upstream 2.3 kbp fragment of PCNA was amplified through PCR and then cloned into the pGL3 basic vector containing a luciferase gene as a reporter. When the luciferase activity was measured in the presence of effector plasmid (pcDNA : c8) expressing murine Hoxc8, the PCNA promoter driven reporter activity was reduced. To confirm whether this reduction is due to the Hoxc8 protein, the siRNA against Hoxc8 (5'-GUA UCA GAC CUU GGA ACU A-3' and 5'-UAG UUC CAA GGU CUG AUA C-3') was prepared. Interestingly enough, siRNA treatment up regulated the luciferase activity which was down regulated by Hoxc8, indicating that Hoxc8 indeed regulates the expression of PCNA, in particular, down regulation in NIN3T3 cells. These results altogether indicate that Hoxc8 might orchestrate the pattern formation by regulating PCNA which is one of the important proteins involved in several processes such as DNA replication and methylation, chromatin remodeling, cell cycle regulation, differentiation, as well as programmed cell death.

• Journal of Plant Biotechnology
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• 제32권3호
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• pp.161-165
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• 2005

Agrobacterium과 자엽절편의 공동배양으로 박과작물인 오이의 형질전환체를 생산하였다. 오이 배양재료는 "은침"의 자엽절편을 사용하였으며, reporter유전자로서 gus유전자와 선발표지로서 bar 또는 nptII유전자로 각각 제작된 pPTN289와 pPTN290벡터를 GV3101, LBA4404, EHA101에 형질전환하여 공동배양하였다. 형질전환빈도는 Agrobacterium의 종류에 따라 현저한 차이가 있었으며, 특히 사용한 균주중 EHA101에서 0.35%로 가장 높았다. 선발배지에서 형성된 오이 식물체중 제초제 저항성 (12개체)과 paromomycin 저항성 (3개체)을 얻었고, 이들 모두 gus양성반응 나타냈다. Southern분석에 의하여 오이 형질전환체의 genome에 gus유전자가 도입되어 있음을 확인 하였다.

• 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.

• Journal of Plant Biotechnology
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• 제35권4호
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• pp.275-280
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• 2008

Agrobacterium공동배양법으로 오이의 기관발생을 통한 형질전환에서 가장 문제점 중 하나는 chimeric 형질전환체의 발생빈도이다. 이러한 문제점을 극복하기 위하여 항생제로서 paromomycin이 첨가된 선발배지에서 "은성" 품종의 배축절편으로부터 체세포배발생을 통한 형질전환시스템을 개발하였다. 배축절편을 pPPTN290발현벡터가 도입된 Agrobacterium 균주 (EHA101)에 30분간 접종한 후 2일간 공동배양 하였고, 선발배지에서 2주 간격으로 5회 계대 배양하면서 항생제 저항성 캘러스 선발, 체세포배발생 및 식물체를 유도하였다. pPPTN290발현벡터의 T-DNA는 reporter유전자로서 Ubi 프로모터에 의해 gus유전자가 발현조절 되도록 그리고 항생제로서 paromomycin에 저항성을 갖는 nptII유전자가 35S 프로모터에 의해 발현되도록 제조하였다. 안정적 형질전환과 빈도는 캘러스의 paromomycin항생제 저항성과 GUS유전자의 발현 여부에 의해 조사하였다. Agrobacterium과 공동배양한 928개의 배축절편에서 paromomycin에 저항성을 갖는 56개의 캘러스 클론을 얻었고, 이중 48개 캘러스 클론 (5.2%)에서 GUS유전자가 안정적으로 발현되고 있음을 확인하였다. 48개의 캘러스 클론중에서 오직 5개의 캘러스 클론으로부터 식물체를 얻어 낮은 빈도 (0.5%)를 나타냈다. 수확한 $T_1$종자에서 GUS양성반응은 gus유전자가 오이 게놈에 안정적으로 도입 및 발현되고 있음을 확인하였다.

• 대한핵의학회지
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• 제38권1호
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• pp.62-73
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• 2004

목적: 단순 헤르페스 제 1형 티미딘 키나제(herpes simplex virus type 1 thymidine kinase gene: HSV1-tk)는 GCV와 함께 유전자치료의 한 방법으로서 가장 활발하게 연구되어왔으며, HSV1-tk 효소에 대한 다양한 기질들이 연구되어서 이를 보고 기질로 한 비침습적인 HSV1-tk 유전자 영상시스템에서 가장 널리 사용되고 있다. 본 연구에서는 보고기질로서 방사성 요오드가 표지된 5-iodovinyl-2-deoxyuridine (IVDU) and 5 -iodovinyl-2-fluoro-2-deoxyuridine (IVFRU)를 보고 기질로 하여 HSV1-tk 유전자 영상시스템에서의 유용성을 확인하고자 하였다. 대상 및 방법 HSV1-tk 유전자 영상을 위하여 HSV1-tk 유전자를 레트로 바이러스 벡터를 이용하여 Morris hepatoma 세포주(MCA-tk)에 이입한 세포주를 제조하였으며, HSV1-tk 유전자의 발현을 확인하기 위하여 Northern blotting과 Western Blotting을 시행하였다. 대조세포주인 MCA와 제조된 MCA-tk 세포주에 방사표지 IVDU와 IVFRU를 이용하여 480분까지 세포내 섭취율을 평가하였으며, 또한 MCA-tk 세포주의 백분율을 증가시키면서 이에 따른 IVDU와 IVFRU의 섭취율을 평가함으로서 섭취율과 세포수와의 상관관계를 평가하였다. 결과: MCA-tk 세포주에서 HSV1-tk의 mRNA의 발현과 HSV1-TK 단백질의 발현을 확인하였다. 방사성 요오드 표지 IVDU와 IVFRU 모두는 MCA 세포주에서는 아주 낮은 섭취율을 나타내었으며, MCA-tk 세포주에서는 모두 증가된 섭취를 보였다. IVDU가 480분에서 IVFRU보다 4배 높은 섭취를 나타내었다(p<0.01). 방사표지 IVDU와 IVFRU 모두에서 MCA-tk의 백분율의 증가에 따라서 직선적인 상관관계($R^2>0.96$)를 나타내었다. 결론: 방사성 요오드 표지 IVDU와 IVFRU는 HSV1-tk유전자가 이입된 간암세포주에서 모두 특이적인 높은 섭취율을 나타내고 직선적인 상관관계가 나타나서 두 기질 모두 HSV1-tk유전자 영상시스템에서 보고 기질로서 유용하게 사용될 수 있을것으로 기대된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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• pp.745-753
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• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• 한국발생생물학회지:발생과생식
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• 제8권1호
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• pp.19-25
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• 2004

Pax6는 진화적으로 잘 보존된 homeobox유전자 그룹의 하나로 배 발생기 동안 시공간적으로 제한되어 발현된다. 이 실험은 말라리아 매개모기인 Anopheles stephemi에서의 Pax6 발현을 서로 다른 분자환경 조건에서 조사해 보기 위해 트랜스포존의 하나인 piggyBac과 Pax6에 결합하는 3xp3-EGFP를 사용한 생식세포 형질전환 방법을 사용하였다. 4개의 형질 전환 계열이 만들어졌고 형질전환율은 6.7%였으며, 도입 유전자는 여러 세대에 걸쳐 안정적으로 발현되었다. 4계열은 3가지의 공간적 발현 형태를 보였으며 이는 트랜스포존 삽입 위치에 따른 enhancing혹은 silencing의 결과로 예상된다. 이 결과를 통해 트랜스포존 piggyBac을 사용한 형질전환 시스템은 일반적인 보고자 유전자 발현 실험에서 다양한 형태의 공간적 발현 결과를 유도하는 매우 효율적인 방법으로 사용될 수 있으리라 예상된다

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1052-1056
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• 2004

A secretion signal sequence-selection vector (pGS40) was constructed based on an $\alpha$-amylase gene lacking a secretion signal and employed for selecting secretion signals from Lactococcus lactis ssp. cremoris LM0230 chromosomal DNA. Six fragments were identified based on their ability to restore $\alpha$-amylase secretion in E. coli, and among these, a fragment, S405, conferred the highest secretion activity (84%) in E. coli. Meanwhile, S407, which conferred poor secretion activity in E. coli, was quite active in L. lactis. The results suggested that the efficiency of a secretion signal depended on the host. All six fragments had an open reading frame (ORF) fused to the reporter gene, and the potential Shine-Dalgamo (SD) sequence and putative promoter sequences were located upstream of the ORF. Deduced amino acid sequences from the six fragments did not show any homology with known secretion signals. However, they contained three distinguished structural features and cleavage sites, commonly found among typical secretion signals. The characterized secretion signals could be useful for the construction of food-grade secretion vectors and gene expression in LAB.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1357-1363
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• 2008

The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.

• 한국잠사곤충학회지
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• 제41권2호
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• pp.102-107
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• 1999

To analysis a promoter strength of Atographa californica nucler polyhedrosis virus (AcNPV) IE1 gene, an immediate viral gene, ${\beta}$-glactosidase gene as a reporter gene was introduced under the control of the IE1 promoter. The restriction fragment containing IE1 promoter and ${\beta}$-galctosidase gene from pAcIE1-gal were inserter into pBacPAK9 to yield transfer vector pAcNPV-IE1-gal. The pAcNPV-IE1-gal was cotransfected with AcNPV genomic DNA BacPAK6 into Sf9 cells to produce recombinant baculovirus AcNPV-IE1-gal. In addition, recombinant bacvulovirus AcNPV-gal, which express ${\beta}$-galac-tosidase under the control of the polyhedrin promoter, was constrer, was constructed to compared with AcNPV-IE1-gal. The recombinant viruses were respectively infected into Sf9 cells and characterized by the virus titer and expression of ${\beta}$-galactoxidase in Sf9 cells. The promoter strength of IE1 and polyhedrin promoters was determined by the amount of ${\beta}$-galactosidase secreted into medium by viral infection. The titer of AcNPV-IE1-Gal determined by plaque assays in Sf9 cells was similar to that of AcNPV-gal. However, expression level of ${\beta}$-galactosidase by AcNPV-IE1-gal was significantly lower than that by AcNPV-gal. In conclusion, promoter strength of IE1 was approximately 25-fold lower than that of polyhedrin.