• BMB Reports
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• 제53권11호
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• pp.565-575
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• 2020

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replication-competent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1346-1352
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• 2005

We have developed a modified gene replacement method using PCR products containing short homologous sequences of 40- to 50-nt. The method required $\lambda$-Red recombination functions provided under the control of a temperature-sensitive CI857 repressor expressed from the $P_{lac}$ promoter in the presence of IPTG on an easily curable helper plasmid. The method promoted the targeted gene replacements in the Escherichia coli chromosome after shifting cultures of the recombinogenic host, which carries the helper plasmid, to $42^{\circ}C$ for 15 min. Since this method employs $\lambda$-Red recombination functions expressed from the easily curable helper plasmid, multiple rounds of gene replacements in the E. coli chromosome would be possible. The procedures described herein are expected to be widely used for metabolic engineering of E. coli and other bacteria.

• 미생물학회지
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• 제26권3호
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• pp.181-187
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• 1988

Using a Tn5-Mob system, pEA9 was characterized as a self-transmissible plasmid carrying a kanamycin resistance marker. The self-transfer frequencies of pEA9 varied greatly depending on pH values. The transfer frequency was about $4\times 10^{-5}$ at pH 5, that was 10 times higher than one at pH 6.5. With a helper plasmid, transfer frequencies were increased about $10^{4}$ times than the frequencies obtained without it.

• 미생물학회지
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• 제29권6호
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• pp.408-415
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• 1991

TOL plsmid size of Flavobacterium odoratum SUB53 was estimated as 83 Md and the optimum concentration of m-toluate degradation by TOL plasmid was 5 mM. $H_{2}$ production by Rhodopseudomonas sphaeroides KCTC1425 was largely dependent on nitrogenase activity and showed the highest at 30 mM malate with 7 mM glutamate as nitrogen source. Nitrogenase activities were inhibited by 0.3 mM $NH_{4}^{+}$ions, to be appeared the decrease of $H_{2}$ production. Conjugation of TOL plasmids from F. odoratum SUB53 and Pseudomonas putida mt-2 to R. sphaeroides showed the optimum at the exponential stage of recipient cells in presence of helper plasmid pRK2013. According to the investigation of catechol-1,2-oxygenase (C-1, 2-O) and catechol-2,3-oxygenase (C-2,3-O) activities of R. sphaeroides C1 (TOL SUB53) and C2 (TOL mt-2), the gene for C-2,3-O is located on TOL plasmid and gene for C-1, 2-O on the chromosome of R. sphaeroides. m-Toluate was biodegraded by TOL plasmid in R. sphaeroides C1 and C2, presumably to be produced $H_{2}$ gas from the secondary metabolites of m-toluate.e.

• KSBB Journal
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• 제16권6호
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• KSBB Journal
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• 제11권1호
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• pp.115-123
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• 1996

zooglan생합성에 필수적인 gene cluster를 clone하기 위해 2종류의 변이주가 분리되었다. Zoogloea ramigera 115는 협막형 다당(주로 zooglan)을 생산한다. 115 균주로 접합시키고 생산물을 용이하게 분리하기 위하여 반복된 원심분리와 선별을 통해 협막을 만들지 않는 slime형 생산균주를 분리하였다. 세포외 다당 생산 능력이 결여된 변이주를 전통적인 transposon(Tn5) 기술을 사용하여 얻었고 달라진 colony 형태와 celluflour결합 성질에 의해 선별하였다. 이들 변이주들은 범용숙주범위 cosmid vector안에 건설된 Z.ramigera 115slime gene library 와 helper plasmid로의 3양친 접합에 의해 보상되었다.

• KSBB Journal
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• 제5권3호
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• pp.247-253
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• 1990

고등식물의 형질전환용 유전자운반체로써 가장 많이 사용되고 있는 Ti-plasmid를 이용해서 당근세포를 형질 전환시키기 위한 연구의 일환으로 Agrobacterium spp를 helper로 이용하여 NPT II gene를 함유하고 있는 binary vector GA472를 당근세포에 삽입시켜 kanamycin에 대해 저항성을 나타내는 세포주를 선발하고자 본 연구를 수행하였다. 국내 토양에서 선발한 A.tumefaciens 2종과 disarmes된 PC2760 그리고 hypervirulent균주인 A281에 tri-parental mating 방법에 의해서 binary vector인 pGA472을 도입하여 transconjungants인 A. tumefaciens c-23-1/pGA472,K29-1/pGA472, PC2760/PGA472 그리고 A281/pGA472를 획득하였다. Transconjungants는 plasmid의 분리, 정제방법에 의해서 추출한 후 0.7% agarose gel 상에서 관찰해 본 결과 4균주 공히 NTPII gene이 삽입된 pGA472와 Ti-plasmid를 함유하고 있는 것을 확인 하였다. 확인된 conjugant와 당근정상조직을 동시배양방법에 의해서 형질전환을 유도한 후 정상조직은 전혀 생존이 되지않은 kanamycin에 대해서 저항을 나타내는 callus를 선발할 수 있었다.

• 미생물학회지
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• 제34권4호
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• pp.236-242
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• 1998

박테리오파지 P2-P4 시스템은 바이러스 조립과정 기작 연구를 위한 뛰어난 실험 소재로 연구되어 왔으나, 이 시스템에 유전자 조작상 필수적인 유용한 플라스미드가 없는 관계로 그의 필요성이 대두되고 있다. 본연구에서는 도움파지가 없을 때 플라스미드 상태로 존재하는 P4 ash8 (sid71)을 시작물질로, 항생제 내성 유전자를 도입하여 선택성을 줄 목적으로 P4 파지 증식에 불필요한 P4 DNA 단편을 pUC4-K 플라스미드의 kmr(kanamycin resistance) 유전자로 치환하여 P4 ash8(sid71) kmr을 생성하였다. 이 플라스미드는 박테리오파지 P2로 induction하여 박테리오파지 P4 상태로 증식시킬 수 있게 그 genome 크기를 조정하였으며, 파지 상태로 전환된 것을 burst size 결정 및 CsCl 부양 균등밀도 편차실험을 통해 입증하였다. 생성된 P4 플라스미드 유도체를 유전자 조작하여 P4의 integrase 기능을 잃은 변이체를 쉽게 만들 수 있었으며, 역시 박테리오파지 P4 상태로 증식시킬 수 있었다. 이러한 변이체 플라스미드의 안정성 실험을 수행하여, P4의 integrase 기능이 지속적인 플라스미드 상태 유지를 위해 필요하다는 것을 보여주었다.

• 한국동물학회지
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• 제38권3호
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• pp.340-347
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• 1995

D. melanogaster를 대상으로 두 종류의 P element construct, 즉 Pc[(ry+)B]와 p[(ry+)$\Delta$SX9]을 사용한 형질전환 실험을 통하여 자율적 및 비자율적인 P element construct간의 vector로써의 효율성을 분석했다. 자율적인 P element 내에 rosy 유전자를 포함하고 있는 Pc[(ry+)B] construct를 진정 M 계통형인 ry506 돌연변이 계통에 주입시켰다. 또한 비자율적인 P element 내에 rosy 유전자를 포함하고 있는 p[(ry+)$\Delta$SX9] construct와 전이효소의 공급원으로써 p-$\Delta$2-3hs$\pi$ helper plasmid를 역시 같은 계통의 ry506 돌연변이 계통에 주입시켰다. Dechorination 방법에 의해 총 1143 embryo를 대상으로 microinjection 한 결과, 모두 35 계통의 형질전환된 정상형의 눈을 가진 계통을 얻었다. P element construct를 주입시킨 전체 1143 embryo 중에서 약 20% 정도가 성체로 부화되었으며, 부화된 개체 가운데 약 40% 정도가 눈 색깔이 거의 정상형과 같은 G0 transformant 로 나타났다. 그러나 생식력을 가진 G0 transformant의 약 40% 만이 G1 transformant로 나타남으로써 모두 35 계통의 transformant를 얻었다. 따라서 G0 단계에서 나타난 형질전환 현상에는 반드시 rosy transformant의 염색체 삽입에 의해서 만이 아니라 세포질내에서도 construct내의 rosy 유전자의 발현이 가능한 것으로 분석되었다. 또한 형질전환율에 있어서는 Pc[(ry+)B]와p[(ry+)$\Delta$SX9] construct 간에 유의한 차가 없는 것으로 나타났다. 이상의 결과로 볼 때, 실험 목적에 따라 자율적인 P element 또는 helper DNA를 이용한 비자율적인 P element를 효과적인 vector 로 선택 이용함으로써 특정 유전자를 곤충집단내로 침투 및 고정시킬 수 있다고 판단된다.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권1호
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• pp.35-38
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• 2007

In this study, ORF 46 of Bombyx mod nucleopolyhedrovirus(Bm46) fused with EGFP was expressed in Bombyx mod cell lines, larvae and pupae by BmNPV Bacmid system. Bm46 and EGFP were cloned into donor plasmid pFastBacHTb, which was transformed to competent DH10B cells containing helper and BmNPV bacmid by site-specific transposition. Recombinant bacmid was used to transfected BmN-4 cells to produce the recombinant baculovirus vBm-Bm46-EGFP. Recombination virus was injected into silkworm larvae and pupae. The expression of the fusion protein was monitored by examining green fluorescence using a fluorescent microscope. Intense fluorescence in cells and silkworm was observed at 4 days post-infection, indicating the Bm46-EGFP fusion gene was expressed successfully.