• 생명과학회지
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• 제16권3호
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• pp.540-545
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• 2006

T-DNA 매개 형질전환은 삽입 돌연변이를 가지는 형질전환 식물체를 만들기 위한 유용한 방법이다. 애기장대의 shoot 발달 과정에서 중요한 역할을 하는 유전자를 확인하기 위해, 프로모터 trap 식물체 라인을 제작하고 분석하였다. 이를 위해 효율적인 프로모터 trap 백터인 pFGL561을 제작하였다. pFGL561은 basta 저항성 유전자, multiple splicing donor acceptor 서열들, 그리고 프로모터가 없는 GFP 리포터 유전자를 포함하고 있다. Agrobacterium 균주인 GV3101에 pFGL561을 도입하고, 이를 매개로 하여 300개의 $T_1$ 프로모터 trap 식물체를 제작하였고, 이들 식물체에서 GFP 발현을 조사하였다. $T_1$ 식물체에서 GFP 발현 비율은 26.7%로 매우 높았고, 이러한 발현은 $T_2$ 식물체에서도 재확인되었다. 한편, 식물의 shoot 발달에 관여하는 주요 유전자를 동정하기 위해서, shoot에서 GFP발현을 보인 19개 $T_1$ 식물체에서 유래한 $T_2$ 식물체를 대상으로 표현형을 조사한 결과, 비정상적인 shoot발달을 보이는 6개 $T_1$ 라인을 확인하였다. 이들 식물체는 shoot발달의 조절 기작 분석에 매우 유용하게 사용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Molecular & Cellular Toxicology
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• 제1권1호
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• pp.32-39
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• 2005

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmentally prevalent xenobiotics that exert complex effects on the biological system and characterized as probably carcinogenic materials. Single cell gel electrophoresis assays were performed in order to evaluate DNA damage occurring in the T-and B lymphocytes, spleens (T/B-cell), bone marrow, and livers of mouse exposed to mixture of PAHs (Benzo(a)pyrene, Benzo(e)pyrene, Fluoranthene, Pyrene) at dose of 400, 800, or 1600 mg/kg body weight for 2 days. DNA damage of the cells purified from mice was increased in dose dependent manner. In the blood cells and organs, DNA damage was also discovered to vary directly with PAHs. Especially T-cells had been damaged more than B-cell. Plasma proteomes were separated by 2-dimensional electrophoresis with pH 4-7 ranges of IPG Dry strips and many proteins showed significant up-and -down expressions with the dose dependent manner. Of these, significant 4 spots were identified using matrix-assisted laser desorption/ionization-time of fight (MALDI-TOF) mass spectrometry. Identified proteins were related to energy metabolism and signal transduction.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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• pp.3791-3794
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• 2012

CD44v, especially splice variants containing exon v6, has been shown to be related closely to development of different tumors. High levels of CD44v6/v7 have been reported to be associated with invasiveness and metastasis of many malignancies. The objective of this study was to detect expression of CD44v6-containing variants in patients with acute promyelocytic leukemia (APL) and evaluate the potential of CD44v6/v7 for risk stratification. Reverse transcription polymerase chain reaction (RT-PCR) followed by PCR product purification, ligation into T vectors and positive clone sequencing were used to detect CD44 v6-containing variant isoforms in 23 APL patients. Real-time quantitative PCR of the CD44v6/v7 gene was performed in patients with APL and in NB4 cells that were treated with all-trans retinoic acid (ATRA) or arsenic trioxide ($As_2O_3$). Sequencing results identified four isoforms (CD44v6/v7, CD44v6/v8/v10, CD44v6/v8/v9/v10, and CD44v6/v7/v8/v9/v10) in bone marrow mononuclear cells of 23 patients with APL. The level of CD44v6/v7 in high-risk cases was significantly higher than those with low-risk. Higher levels of CD44v6/v7 were found in three patients with central nervous system relapse than in other patients inthe same risk group. Furthermore, in contrast to ATRA, only $As_2O_3$ could significantly down-regulate CD44v6/v7 expression in NB4 cells. Our data suggest that CD44v6/v7 expression may be a prognostic indicator for APL.

• 한국가금학회지
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• 제36권2호
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• pp.177-186
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• 2009

형질전환 동물에 있어서 외래 유전자의 조절되지 않은 과다 발현은 생리적인 부작용이나 독성을 나타내게 된다. 본 연구에서는 이러한 문제를 해결하기 위하여 외래 유전자 발현 조절 system인 tetracycline-inducible expression system(Tet system)을 도입하였다. 그러나 종래의 Tet system을 이용한 유전자 발현 조절은 system 자체의 구성 요소로 인한 미미한 leaky 현상 때문에 완벽하게 이루어지지 않는다. 본 연구에서는 보다 완벽한 외래 유전자의 발현 조절 system을 구축하기 위하여 rtTA 대신 진일보한 형태의 $rtTA2^SM2$를 도입하고 TRE 부분을 TRE-tight로 대체하였다. 확립된 retrovirus vector system을 이용하여 다양한 표적세포와 형질전환 닭으로부터 혈소판 생산의 일차적인 조절자이며, 조혈간세포의 생존과 증식에 있어서 매우 중요한 역할을 하는 human thrombopoietin(hTPO)를 생산하고자 하였다. In vitro 상의 연구에서, CEF 세포에서 발현되는 hTPO가 가장 높은 발현량과 발현 유도율을 나타내었으며, 상업적으로 판매되고 있는 hTPO나 다른 표적세포에서 생산된 hTPO에 비해 생물학적 활성이 가장 높은 것으로 확인되었다. 고농도로 농축한 재조합 retrovirus를 stage X단계의 계란의 배반엽 층에 미세주입하여 대리 난각 방법으로 배양한 결과, 미세주입한 138개의 계란 중 21일 후에 15개의 계란에서 병아리가 부화하였으며, 그 중 8마리가 형질전환 개체로 확인되었다. 이 형질전환 닭은 사료 1 g 당 0.5 mg의 doxycycline을 첨가하여 2주간 식이하였으며, 그 후 혈액을 채취하여 hTPO 농도를 측정한 결과 200ng/mL로 확인되었다. 또한 형질전환 개체 중 수컷의 정자에서 hTPO 유전자의 존재를 확인함으로써 germLine transmission의 가능성을 입증하였다. 이상의 연구 결과는 사람의 cytokine 단백질의 대량생산을 위한 생체반응기로서의 형질전환 닭의 생산 가능성을 제시한 데 의의가 있다.

• 건축역사연구
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• 제32권2호
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• pp.37-48
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• 2023

A literature study was conducted on the management of the pavilion of the royal tomb in the late Joseon Dynasty, focusing on "Gakneung Suri Deungnok(Records relating to the repair of royal tombs)". This study analyzed the royal tomb management system, organized the types of damage identified in the building, and examined how the damage status was recorded by type. In the above, the records related to the 1675~1713 repair of three JeongJaGak(Geonwonneung, Sungneung, and Mokneung), which are registered as state-designated cultural properties, are summarized in three aspects: management system, damage status, and expression words. The results of the study are as follows. First, the royal tomb pavilion was regularly inspected by Observator(觀察使) in spring and autumn, and Surunggwan(守陵官) every 5th, and Servant(守僕) regularly inspected every day and night, and also inspected and reported emergency cases of natural disasters or unexpected damage. Second, the damage status of each building was continuously observed and reported for the continuous maintenance of the buildings in the royal tomb. A total of 75 records of damage to the three royal tombs' pavilion were found to have been most frequently inspected, including 19 cases (25.3%), 14 cases (18.7%), 23 cases (30.7%) of the roof, and 19 cases (25.3%) of the roof. Third, the expression of the damage status is confirmed in various ways, such as separation, separation, burst, damage, excitation, moisture, leakage, and exfoliation. Among them, the main damage records were confirmed due to the separation of the base from the peeling, the furniture, cracks, leaks, leaks in the roof, and the collapse of the roof was able to check the damage records.

• Reproductive and Developmental Biology
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• 제41권1호
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• pp.7-16
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• 2017

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the ${\beta}-casein$ gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5' arm region and 1.8 kb or 0.64 kb of 3' arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5' terminal of endostatin gene and inserted into exon 7 of the ${\beta}-casein$ gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3' arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine ${\beta}-casein$ gene in the mammary gland.

• BMB Reports
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• 제34권3호
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• pp.230-236
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• 2001

dTDP-rhamnose is synthesized from dTTP and glucose-1-phosphate by four enzymatic steps in the gram-negative bacteria. By using a homologous PCR product, a gene cluster encoding four genes (rfbA, rfbB, rfbC, rfbD) involved in L-rhamnose biosynthesis by Acinetobacter calcoaceticus was isolated and sequenced. The four genes were clustered on the biosynthetic operon in the order of rfbB, D, A, C. A gene, rfbA, encoding glucose-l-phosphate thymidylyltransferase (RfbA), was cloned from A. calcoaceticus pathogenic and encapsulated in the gram-negative bacterium. This enzyme catalyzes the formation of dTDP-D-glucose From $\alpha$-D-glucose-1-phosphate and dTTP.RfbA was amplified by PCR and inserted into the $T_7$ expression system. The activity of RfbA was determined by the capillary electrophoresis. The $K_m$ values for dTTP and $\alpha$-D-glucose-1-phosphate were calculated to be 1.27 mM and 0.80 mM, respectively by using the Line-Weaver Burk plot. RfbA is inactivated by diethylpyrocarbonate.

• 대한세포병리학회지
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• 제7권1호
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• pp.38-43
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• 1996

Epstein-Barr virus(EBV) is associated with a wide spectrum of benign and malignant disorders including leukoplakia, Hodgkln's lymphoma, central nervous system lymphoma, peripheral T cell lymphoma and nasopharyngeal undifferentiated carcinoma. There are several distinctive aspects of biology of the virus that are important in investigation of virus in clinical specimens. The abundant expression of the EBER mRNA transcripts makes possible the sensitive detection of latent expression in EBV-associated tumors. Although there has been a dramatic increased interest in the direct characterization of EBV in clinical specimens, there have been few studios about the effective and reliable positive controls in performing in situ hybridization technique for EBV, especially on paraffin-em bedded tissue. We applied Burkitts lymphoma ceil line as positive control in EBV in situ hydridization using Oncor Kit. The cell block of Burkitt lymphoma cell line(CCL85 EB-3) showed strong and specific positivity for EBER in situ in nuclei of EBV infected cells.

• Journal of Plant Biotechnology
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• 제45권1호
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• pp.30-35
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• 2018

We established an in vitro system to investigate transcription levels of the RsMYB1 gene expressed in T2 20-day-old transgenic Petunia plants (three independent lines: PhRs1, PhRs2, and PhRs3), and the association between those transcription levels and anthocyanin production at various pH values (3.0 to 8.0) for a period of 10 days. All the lines treated with pH 5.0-7.0 exhibited increased anthocyanin content and delays in growth compared to the wild-type (WT) seedlings. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed that the enhancement of anthocyanin production in the transgenic lines was due to the upregulation of RsMYB1 transcription at various pH values. The results suggest that pH value can control expression of RsMYB1 which is associated with anthocyanin production.