• BMB Reports
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• 제33권6호
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• pp.476-482
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• 2000

In this paper, we report a new cationic lipid composed of L-lysinamide and cholesterol as a potent gene delivery vector. $3{\beta}$[L-Lysinamide-carbamoyl] cholesterol could self-assemble with plasmid DNA forming discrete lipoplexes. From atomic force microscopic images of the complexes, the size distribution was observed to range from 100 to 150 nm in diameter. The transfection efficiency of this amphiphile on different cell lines was evaluated as a micellar solution in the absence of the fusogenic helper lipid, dioleoyl phosphatidyletbanolamine (DOPE). Transfection experiments were performed as a function of charge ratio (lipid/DNA) and transfection time. Cytotoxicity and in vitro transfection efficiency of the amphiphile was demonstrated and compared with those of commercially available Lipofectin and polyethylenimine (PEI).

• Current Research on Agriculture and Life Sciences
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• 제5권
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• pp.52-58
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• 1987

효모(酵母)와 대장균(大腸菌)의 셔틀 벡터인 YIp5, YRp7, YEp13플라스미드를 S. cerevisiae DBY747을 수용세포(受容細胞)로 하여 원형질체 방법(方法)에 의해 형질전환(形質轉換)시킨 결과(結果), YIp5플라스미드는 형질전환체가 나타나지 않았으며, YEp13플라스미드는 DNA $10{\mu}g$ 당(當) $1.2{\times}10^3$, YRp7은 $1.0{\times}10^2$ 개(個)의 형질전환체를 얻었다. YIp5플라스미드와 YEp13플라스미드, 그리고 YIp5플라스미드와 YRp7플라스미드를 환상(環狀)플라스미드 상태(狀態)로 동시형질전환(同時形質轉換) 시켰을 때 각각 DNA $10{\mu}g$ 당(當) 210 및 95개(個)의 형질전환체를 얻었으며, 동일(同一) 부위(部位) 또는 다른 부위(部位)를 절단(切斷)한 선상(線狀)플라스크를 앞서와 같이 동시형질전환(同時形質轉換)시켰을때, 서로 비슷하게 DNA $10{\mu}g$당(當) 15~85개(個)의 형질전환체를 얻을 수 있었다. 이러한 결과(結果)에서 볼때 수용세포(受容細胞)를 S. cerevisiae DBY747로한 동시형질전환(同時形質轉換)에서 환상(環狀)플라스미드간(間)의 형질전환(形質轉換)이 선상(線狀)플라스미드간(間)의 형질전환(形質轉換)보다 빈도(頻度)가 높으며 선상(線狀)플라스미드간(間)의 형질전환(形質轉換)에서 미단(未端) 부위(部位)의 상이(相異)함이 큰 영향을 주지 않는 것으로 나타났다. 형질전환체의 안정성(安定性)에 있어서는 YIp5플라스미드와 YEp13플라스미드간(間)의 형질전환체가 YIp5플라스미드와 YRp7플라스미드간(間) 형질전환체에 비(比)해 안정(安定)하게 나타났는데, 이는 stringent control을 받는 ARS유전자(遺傳子)를 가진 YRp7플라스미드보다 $2{\mu}m$ DNA 복제(複製) 기점(起點)을 가진 YEp13플라스미드의 복제수(copy number)가 많기 때문인 것으로 생각된다.

• 한국산림과학회지
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• 제77권2호
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• pp.199-207
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• 1988

포플러류(類)에 대(對)한 유용유전자(有用遺傳子) 삽입에 관(關)한 기초(基礎) 연구(研究)로서, 북미(北美)의 자연잡종(自然雜種) 포플러, P. alba ${\times}$ P. grandidentata 3 클론을 대상으로 Agrobacterium tumefaciens A281과 A348 strain의 감염력을 조사(調査)하였다. Kanamycin 저항성(低抗性)의 neomycin phosphotransferase (NPT-II) 유전자(遺傳子)를 가진 Agrobacterium binary pGA 472 vector와 이들 조직배양(組織培養)된 세 클론의 잎조각을 함께 배양(培養)하여, 형질전환(形質轉換)된 부위(部位)를 선발(選拔)하기 위해서 kanamycin sulfate의 적정농도(適正濃度)를 조사(調査)하였다. A281과 A348 strain 중(中) A281 strain이 가지고 있는 pTiBo542가 binary vector system의 helper plasmid로서의 역할에 가장 적합한 것으로 나타났다. 비교적 저농도(低濃度)(10mg/l)의 kanamycin sulfate가 잎조각배양으로 부터 식물체(植物體) 유도를 억제하였다. Kanamycin 저항성(低抗性) 유전자(遺傳子)가 내포된 Agrobacterium binary vector와 잎조각을 함께 배양(培養)한 후(後) kanamycin에 대한 저항성(低抗性)을 가진 callus가 kanamycin(60mg/l)이 들어있는 식물체 유도배지에서 선발(選拔)되었다. Kanamycin 저항성(低抗性) 유전자(遺傳子)에 의해 형질전환된 이들 kanamycin 저항성(低抗性) callus와 정상적인 비교 callus를 kanamycin이 들어있는 식물체(植物體) 유도배지에서 배양(培養)했을때 정상적인 비교 callus는 50mg/l의 kanamycin 농도(濃度)에서 생장(生長)이 억제된 반면, 형질전환(形質轉換)된 kanamycin 저항성(低抗性) callus는 200mg/l의 kanamycin 농도(濃度)에서도 생장(生長)을 계속하였다.

• Archives of Pharmacal Research
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• 제21권5호
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• pp.537-542
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• 1998

to more effectively drive immune responses toward antigen-specific T helper type 2 (Th2) cell-mediated responses, we constructed a mammalian expression vetor (oPVA/IL4) carrying a fused gene in which the ovalbumin (OVA) cDNA was covalently linked to murine interleukin-4 (IL-4) cDNA. A biologically active OVA/IL4 DNA, as demonstrated by Wes tern blotting and cytokine bioassay. In tramuscular injection of BALB/c mice with the pOVA/IL4 DNA increased both the production of OVA-specific IL-4 by CD$4^{+}$ T cells and the ratio of anti-OVA lgG1 to anti-OVA lgG2a isotypes, while the injection with the pOVA DNA alone, or with the mixture of the pOVA and pIL4 DNA did no or little increase. furthermore, the OVA-specific, Th2 cell-mediated immune responses were significantly enhanced by multiple injections with the pOVA/IL4 DNA. These studies indicate that the direct linkage of an OVA gene to an IL-4 gene in the expression plasmid confines the effects of IL-4 to the OVA-specific cells, efficiently driving the immune response toward OVA-specific, Th2 cell-mediated responses.

• Journal of Microbiology
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• 제44권5호
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• pp.530-536
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• 2006

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasm ids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size bead, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.

• 한국식물병리학회지
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• 제10권3호
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• pp.151-156
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• 1994

Pseudomonas fluorescens produces the antibiotic, 2,4-diacetylphloroglucinol (Phl), which promotes plant growth by inhibiting bacteria and fungi. Cosmids (genomic library) were mobilized into Phl-nonproducing mutants through the triparental matings with pRK2013 as the helper plasmid at the frequency of 8.37$\times$10-4. Complemented mutants that showed antibiotic activity were selected among about 2,000 transconjugants. The complemented mutants were confirmed by acquired drug resistances (kanamycin and tetracycline). The antibiotic substances of wild type and complemented mutants showed the most excellent anti-bacterial activity. Inhibitory effects of complemented P. fluorescens against phytopathogenic fungi were equal to the parental strain. Complemented mutant and wild type of P. fluorescens were causal microbes of fungal morphological abnormalities. Complemented mutants in potato dextrose agar supplemented with bromothymol blue also showed restoration of glucose utilization as wild type. Plasmids of complemented mutants were isolated from transconjugant sand transformed into competent cells of E. coli DH5$\alpha$. The plamid DNA was reisolated from transformed E. coli DH5$\alpha$.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1279-1287
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• 2012

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, $10^{-5}-10^{-3}\;recipient^{-1}$). pSTE33T showed lower efficiency ($10^{-7}-10^{-6}\;recipient^{-1}$) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

• Bulletin of the Korean Chemical Society
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• 제34권3호
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• pp.931-935
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• 2013

Lipid events in liposome-mediated transfection (lipofection) are largely unknown. Here we studied whether phospholipase D (PLD), an important enzyme responsible for phospholipid breakdown, was affected during lipofection of HepG2 cells with a luciferase plasmid. Synthetic cholesterol (Chol) derivatives, including $3{\beta}$[L-ornithinamide-carbamoyl]Chol, [polyamidoamine-carbamoyl]Chol and $3{\beta}$[N-(N',N'-dimethylaminoethane)-carbamoyl]Chol, and a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride were mixed with a helper lipid dioleoylphosphatidylethanolamine to form respective cationic liposomes. All cationic liposomes were found to stimulate PLD. Although orders of magnitude effects of the cationic liposomes on PLD stimulation did not consistently match those on cytotoxicity and luciferase expression, a causal relationship between PLD activation and cytotoxic effect was remarkable. PLD stimulation by the cationic liposomes was likely due to their amphiphilic characters, leading to membrane perturbation, as supported by similar results obtained with other membrane-perturbing chemicals such as oleate, melittin, and digitonin. Our results suggest that lipofection induces cellular lipid changes such as a PLD-driven phospholipid turnover.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.174-182
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• 1992

The hemagglutinin-esterase glycoprotein (HE) gene of bovine coronavirus, coupled with a simian virus 40 early promoter and polyadenylation signal, was inserted into a human adenovirus transfer vector. The transfer vector was used to co-transfect 293 cells along with adenovirus genomic DNA. The hemagglutinin-esterase transcription unit was rescued into the adenovirus genome by homologous in vivo DNA recombination between the vector plasmid DNA and the adenovirus genomic DNA, and a recombinant adenovirus was isolated by several rounds of plaque assays. Thus the recombinant adenovirus carries the hemagglutinin-esterase gene in the early transcription region 3 (E3) of the adenovirus genome in the parallel orientation to the E3 transcription. The recombinant adenovirus synthesized the HE polypeptide in HeLa cells as demonstrated by immunoprecipitation with anti-coronavirus rabbit antisera. The recombinant HE polypeptide could be labelled by $[^3H]$glucosamine, demonstrating that the recombinant HE was glycosylated. Cells expressing the HE polypeptide exhibited hemadsorption activity when incubated with mouse erythrocytes. The HE was transported to the plasma membrane as shown by the cell surface immunofluorescence, indicating that the recombinant HE polypeptide retained its biological activities. Potential for the use of infectious recombinant adenovirus as a live virus-vectored vaccine candidate for bovine coronavirus disease is discussed.

• 대한수의학회지
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• 제57권1호
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• pp.9-15
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• 2017

To construct a novel vaccine candidate against bovine enterotoxigenic Escherichia coli (ETEC), FanC, the major subunit of K99 fimbriae adhesion, was inserted into secretion plasmid pYA3560 containing a ${\beta}-lactamase$ secretion system. This was then transformed into ${\Delta}asd$ ${\Delta}crp$ Salmonella (S.) Typhimurium and designated as JOL950. Secretion of recombinant fanC fimbrial antigens was confirmed by immunoblot analysis. Groups of mice were inoculated with single or double doses of JOL950. Another group was used as a negative control. Compared to control mice, all immunized mice had significantly higher levels (p < 0.05) of serum immunoglobulin (Ig)G, and secretory IgA against FanC. The IgG2a and IgG1 titer assays revealed that immunization highly induced IgG2a compared to that of IgG1, indicating that T helper-1- related cell-mediated immune responses may be elicited by JOL950. The results show that both systemic and mucosal immunities against selected fimbrial antigens of bovine ETEC expressed by a live attenuated S. Typhimurium strain are prominently produced in mice immunized with JOL950 via an oral route.