• Asian-Australasian Journal of Animal Sciences
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• 제17권1호
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• pp.27-32
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• 2004

Using MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein), we tried to make transgenic chickens carrying the transferred genes in their chromosomes. Twenty one days after virus injection beneath the blastoderms of unincubated chicken embryos (stage Ⅹ, at laying), DNA isolated from the hatched chicks were analyzed by PCR with two sets of primers specific for EGFP (enhanced green fluorescence protein) gene or $Neo^R$ (E. coli neomycin resistant) gene. Among sixty-seven embryos injected with retrovirus, four of them were identified to carry the EGFP genes in their genomes. Remarkably, one transgenic chick showed presence of the retrovirus vector sequences in all organs differentiated from one of endoderm, mesoderm, and ectoderm. Expression of EGFP gene was not detected, however, the stable germ line transmission of transgene was verified in spermatozoa from the founder chicken and 50% of $F_1$ progenies.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.109-115
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• 2002

Gene therapy is to treat and cure diseases by an introduction of therapeutic genes in defective cells or tissues of human body. Gene delivery system, gene expression system, and therapeutic gene are three core elements for gene therapy. The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for therapeutic outcome of gene delivery. Because it can be used in common for the treatment and cure of various diseases, gene delivery system is the most important core element for a successful gene therapy. Viruses are naturally evolved to transfer their genomes into host cells efficiently. This ability has made vectorologists exploit viruses as attractive vehicles for the delivery of therapeutic genes. Viral vectors based on adenovirus (Ad) and adeno-associated virus (AAV) have been often used for gene delivery in laboratory. Ad and AAV vectors derived from human DNA viruses differ greatly in their life cycle, expression level and duration of transgenes, immunogenicity, and vector preparation. Both vectors can be used as effective tools for gene therapy and more recently in functional genomics. Here, the characteristics of Ad and AAV vectors are discussed.

• 미생물학회지
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• 제38권2호
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• pp.86-95
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• 2002

최근 인간을 비롯한 다양한 생명체의 genome project연구결과 밝혀진 유전자들의 염기서열을 토대로, 생명체 구성성분의 실질적 인 역할을 하는 단배질의 기능을 밝히는 proteomics에 관한 연구의 필요성 이 대두되고 있다. 따라서, 이 연구는 post-genomics시대에 다양한 종류의 단백질 기능과 상호작용의 기초연구에 필수적 인 새로운 포유동물세포 유전자 발현벡터를 RNA 바이러스인 소설사성 바이러스(Bovine Viral Diarrhea Virus)의 infectious CDNA molecular clone을 이용하여 개발하였다. 먼저 BVDV의 infectious CDNA molecular clone (pNADLclns-)을 이용하여 puromycin 항생제에 저항성을 나타내는 puromycin acetyltransferase (pac) 유전자를 삽입하여 recombinant full-length infectious CDNA clone을 합성하였다. 합성된 recombinant CDNA clone을 주형으로 T7 RNA polymerase를 사용하여 in vitro transcribed full-length viral RNA를 합성하였다. 합성된 viral RNA의 자가복제 여부는 MDBK세포에 transfection시킨 후, $^{32}P$ 로 metabolically label함으로써 확인하였다. 또한, transfection된 세포에서의 바이러스 단백질 발현여부는 바이러스에 특이적으로 반응하는 anti-NS3 단클론항체를 사용하여 분석하였다. 또한, infectious CDNA clone을 응용하여 새로운 포유동물세포유전자 발현벡터의 개발을 위해서, 먼저 바이러스의 구조단백질이 바이러스의 자가복제에 필수적인 지를 평가하였다. 실험결과, 각각의 구조단백질 유전자를 deletion한 recombinant cDNA clone으로부터 합성된 viral RMA의 자가복제여부는pac유전자의 발현여부로 recombinant cDNA clone으로부터 합성된 recombinant viral RMA를 MDBK 세포에 transfection시킨 후, puromycin으로 selection함으로써 할 수 있었다. Deletion실험결과, 각각의 구조단백질 capsid및 E0, El, E2는 바이러스의 자가복제에 영향을 기치지 않음을 알 수 있었다. 이와 더불어, 바이러스의 모든 구조단백질을 함께deletion하였을 경우에도 자가복제에는 영향을 기치지 않는 것을 합성된 viral replicon을 이용한 실험에서 알 수 있었다. 이렇게 합성된 BVDV의 replicon을 사용하여 포유동물의 발현벡터로써 사용할수 있는 지의 여부를 분석하기 위해서 pac유전자 이외에 luciferase유전자를 사용하여 MDBK및 HeLa, BHK세포에서의 단백질 발현정도를 시간 별로 분석한 결과, BVDV의 replicon을 다양한 종류의 유전자 발현벡터로사용할 수 있음을 알 수 있었다. 그러므로, RNA바이러스의 하나인 BVDV의 viral replicon을 이용하여 다양한 종류의 포유동물 세포에 유전자 발현벡터로써 사용할 수 있음으로 post-genomics시대에 다양한 종류의 단백질 기능연구에 맡은 도움이 되리라 기대한다.

• Journal of Plant Biotechnology
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• 제43권4호
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• pp.432-437
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• 2016

Interleukin-11 (IL-11) is a cytokine that plays a key regulatory role in the immune system. Recombinant human IL-11 (rhIL-11) exerts a preventative effect against apoptotic cell death and inhibits preadipocyte differentiation. IL-11 also is used to stimulate the bone marrow to produce platelets in order to prevent low platelets that may be caused by chemotherapy. Unfortunately, the high production cost of IL-11 associated. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-11. Production of rhIL-11 proteins in whole-plant expression system will be more economical when compared to the current E. coli based expression system. The human rhIL-11 gene was codon optimized to maximize plant host system expression. IL-11 expression vector under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into tobacco by Agrobacterium-mediated transformation. The 5'-leader sequence (called ${\Omega}$) of tobacco mosaic virus (TMV) as a translational enhancer was added to construct. Transgenic tobacco plants expressing various levels of rhIL-11 protein were generated. Western blotting of the stably transformed lines demonstrated accumulation of the appropriately sized rhIL-11 protein in leaves. This research demonstrated the efficacy of using tobacco as an expression system for the production of rhIL-11.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 추계 학술대회
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• pp.31-50
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• 2000

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene e(pression. However, we have now developed a series of reroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors the intron and exon sequences from heterologous cellular or viral genes are present, When compared to the well blown MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences

• 대한수의학회지
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• 제53권4호
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• pp.217-224
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• 2013

Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-$S_{0.7}$) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-$S_{0.7}$ gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-$S_{0.7}$ protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-$S_{0.7}$ antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

• 식물조직배양학회지
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• 제27권3호
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• pp.169-173
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• 2000

Amaranthus hypochondriacus의 저장 단백질을 encoding 하는 AmA1 유전자를 RT-PCR 방법을 이용하여 분리하고 특성화하였다. AmAl 유전자를 담배에 형질전환 시키기 위해 CaMV 35S promoter와 3'NOS를 가지고 있는 식물 발현 vector에 subcloning 하고 이 재조합 vector를 이용하여 Agrobacterium-mediated형질전환 방법을 이용하여 담배에 도입시켰다 신초는 0.1 mg/L NAA, 1.0 mg/L BA, 100 mg/L kanamycin 그리고 250 mg/L cefotaxime이 첨가된 MS 선발 배지에서 선발했고, 선발된 신초는 식물 생장 조절제를 첨가 하지 않고 200 mg/L kanamycin과 250 mg/L cefotaxime이 첨가된 MS배지에서 뿌리를 유도하였다. 선발된 담배의 게놈내의 AmA1 유전자의 존재는 PCR 방법과 hybridization을 이용하여 확인되었고, AmA1 유전자의 발현은 RT-PCR방법과 Southern blot hybridization을 사용하여 확인되었다.

• Molecules and Cells
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• 제42권5호
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• pp.418-425
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• 2019

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

• BMB Reports
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• 제36권3호
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• pp.275-281
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• 2003

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.