• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.153-159
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• 2008

The limitations in current technology for generating transgenic animals, such as the time and the expense, hampered its extensive use in recombinant protein production for therapeutic purpose. In this report, we present a simple and less expensive alternative by directly infusing a recombinant adenovirus vector carrying human lactoferrin cDNA into rabbit mammary glands. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. An 80-kDa protein was visualized after viral vector infection. With this method, we obtained a high level of expressed human lactoferrin of up to 2.3mg/ml in the milk. Taken together, the method is useful for the transient high-level expression recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest.

• BMB Reports
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• 제42권1호
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• pp.59-64
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• 2009

Hepatitis B virus (HBV) infection is highly prevalent worldwide. The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by $87{\pm}4$, $80.3{\pm}2.6$ and $86.2{\pm}7%$ respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance.

• 미생물학회지
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• 제29권2호
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• pp.75-79
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• 1991

The yeast L-A virus (4.6 kb dsRNA genome) encodes the major coat protein and a "gag-pol" fusion minor coat protein that separately encapsidate itself and $M_{1}$, a 1.8 kb dsRNA satellite virus encoding a secreted protein toxin (the killer toxin). The teast chromosomal SKI genes prevent viral cytopathology by lowering the virus copy number. Thus, $ski^{-}$ mutants are ts and cs for growth. We transformed a ski2-2 virus-infested mutant with a yeast bank in a high copy cloning vector and selected the rare healthy transformants for analysis. One type of transformant segregated M-O L-A-O cells with high frequency. Elimination of the DNA clone from the ski2-2 strain eliminated this phinotype and introduction of the DNA clone recovered from such transformants into the parent ski2-2 strain, or into ski3 or ski6 mutants gave the same phenotype. This killer-curing phenotype was due to the curing of the helper L-A dsRNA virus. The 6.5 kb insert only had this activity when carried on a high copy vector and in $ski^{-}$ cells (not in $SKI^{+}$ cells). This 6.5 kb insert acts as a mutagen on L-A dsRNA producing a high rate of deletion mutations.mutations.

• The Plant Pathology Journal
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• 제22권4호
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• pp.386-390
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• 2006

Extensive research of the Potato virus X(PVX) has been performed in in vitro transcription system using the bacteriophage T7 promoter. We constructed an efficient T-DNA based binary vector, pSNU1, and modified vectors carrying PVX replicons. The suitability of the construct to transiently express PVX RNA using Agrobacterium tumefaciens was tested by analysis of infectivity in plants. The expressed PVX RNA was infectous and systemically spread in three plant species including Nicotiana benthamiana, N. tabacum cv. Xanthi-nc, and Capsicum annuum cv. Chilsungcho. The PVX full length construct, pSPVXp31, was caused severe mosaic symptoms on N. benthamiana, severe necrotic lesions on C. annuum while milder symptoms and delayed mosaic symptoms were appeared on the systemic leaves on N. tabaccum. RT-PCR analysis confirmed the presence of PVX RNAs on both inoculated and systemic leaves in all three plant species tested. Our results indicated that PVX replicons were efficiently expressed PVX RNA in at least three tested species. Further investigation win be needed to elucidate the mechanism of PVX replication, translation, movement and assembly/disassembly processes.

• 한국연초학회지
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• 제19권1호
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• pp.11-17
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• 1997

A flue-cured tobacco variety (Nicotiana tabacum cv. Wisconsin) was used for Plant transformation with the complementary DNA (cDNA) of potato virus Y-necrosis strain (PVY-VN) replicase gone (Nb) which was synthesized through reverse-transcription Primed with oligo(dT) and Polymerization using RNase H-digested template. The cDNA was cloned into Plant expression vector Plasmid (PMBP2), and introduced into tobacco plants by co-culturing tobacco leaf disks with Agrobacterium tumefaciens LBA4404 containing the plasmid before Plant regeneration. Eight Plants, in which the inserted cDNA fragment was detected by Polymerase chain reaction (PCR), out of 70 putative transformants inserted with sense-oriented Mb cDNA showed no symptom at 3 weeks after inoculation, while the other 62 plants, and all plants with vector gone only and antisense-oriented NIb cDNA had susceptible vein-necrosis symptoms. However, only 2 of the 8 resistant plants were highly resistant, which remained symptomless up to 10 weeks after inoculation. Among the first progenies (T1) from self-fertilized seeds of the two resistant transgenic plants, less than 10 % of 71 plants appeared highly resistant (with no symptom), 70% moderately resistant (with mild symptoms on 1 - 2 leaves), and about 20% susceptible (with susceptible symptoms on 3 or more leaves) at 3 weeks after inoculation. These results suggest that the PVY resistance was inherited in the 71 generation. Key words : potato virus Y. viral replicase gene, transgenic tobacco Plants, resistance.

• Molecules and Cells
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• 제40권8호
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• pp.598-605
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• 2017

Human mesenchymal stem cells (MSCs) are currently being evaluated as a cell-based therapy for tissue injury and degenerative diseases. Recently, several methods have been suggested to further enhance the therapeutic functions of MSCs, including genetic modifications with tissue- and/or diseasespecific genes. The objective of this study was to examine the efficiency and stability of transduction using an adenoviral vector in human MSCs. Additionally, we aimed to assess the effects of transduction on the proliferation and multipotency of MSCs. The results indicate that MSCs can be transduced by adenoviruses in vitro, but high viral titers are necessary to achieve high efficiency. In addition, transduction at a higher multiplicity of infection (MOI) was associated with attenuated proliferation and senescence-like morphology. Furthermore, transduced MSCs showed a diminished capacity for adipogenic differentiation while retaining their potential to differentiate into osteocytes and chondrocytes. This work could contribute significantly to clinical trials of MSCs modified with therapeutic genes.

• 대한바이러스학회지
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• 제26권1호
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• pp.77-90
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• 1996

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

• Bulletin of the Korean Chemical Society
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• 제32권5호
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• pp.1569-1574
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• 2011

The presence of replication-competent adenovirus (RCA) subpopulations in adenoviral vector products raises a variety of safety issues for development of therapies based on gene therapy. To analyze the differing effects of adenoviral vector and RCA in vivo, we examined alterations in amino acids (AAs) using rat plasma following injection of ${\beta}$-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA. Plasma AAs were examined by gas chromatography-mass spectrometry. A total of 16 AAs were positively measured. In the rAdLacZ group compared to the control group, the level of aspartic acid was significantly increased (Student's t-test), while the level of glutamic acid was significantly reduced. Additionally, in the RCA group compared to the control group, the level of four AAs, valine, leucine, and isoleucine as branched-chain amino acids, and proline were significantly increased, whereas the levels of three AAs, glycine, threonine, and glutamic acid were significantly reduced. Altered plasma free AA metabolic patterns in rAdLacZ and RCA groups, compared with the control group, may explain the disturbance of AA metabolism related to viral infection.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.62-62
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• 2001

Recently, production of transgenic animal by nuclear transfer has been known as a useful method. The production of cloned offspring derived from nuclear transfer depends upon a variety of factors such as species, donor cells type and cell cycle, and source of recipient ova. Therefore, we attempted a different transgenic methods using follicular granulosa cells (GCs). In general, ovulated GCs undergoes lutenization and transformation in vitro which might defective effects on developmental potential. In order to avoid the GCs transformation in vitro culture system, we introduced a direct injection of retrovirus into the follicles and then collected them mechanically from ovaries of 6-8 week-old ICR mice. Retrovirus vector constructed with pLN $\beta$ EGFP was injected into the follicles. The follicles are cultured in $\alpha$ -MEM supplemented with human FSH, LH and ITS in Costar Transwell dish for 4 days. Survival rate of virus injected follicles was 52.1% (12/23) and expression rate of EGPP gene was 33.3% (4/12). In this study, we found GCs performed transgenesis in our culture system. In addition, the GCs in follicle may be developed in vivo like environment rather than in vitro environment. Thus, the use of GCs as donor cells may be useful in the nuclear transfer for cloning of genetic modification. Therefore, these results suggest that follicular GCs can be transfected by viral vector during folliculogenesis in vitro.

• 미생물학회지
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• 제39권2호
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• pp.118-122
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• 2003

바이러스 조립 과정 기작 연구를 위한 좋은 재료인 박데리오파아지 P2-P4시스템에 이용될 벡터 플라스미드를 개발하기 위하여, P4 ash8 sid71을 출발 물질로 삼아 새로운 P4 유도체 벡터를 조성하였다. 유전자 재조합 기법을 써서 쉽게 선택 가능한 tetracyclin내성 유전자(tetR)를 도입하고 플라스미드P 4의 크기를 조절하였다. 이를 통해 얻어진 P4 ash8(sid7l) tetR은 12.09 kb의 크기를 가지며, 필요할 때 P2로 induction하면 생물학적 활성을 가지는 박데리오파아지로 전환 가능하였다. 전환된 파아지의 burst size를 결정하고, CsCi 부양균등밀도 편차실험을 수행하였다. 균등밀도 실험 분포도에서 P2크기 파아지 머리의 packaging 상한을 추정할 수 있었다.