• Archives of Pharmacal Research
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• 제16권2호
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• pp.155-157
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• 1993

DNA transfection conditions were investigated by calcium phosphate-DNA co-precipitation in SK-N-BE(2)C human neuroblastoma cells. The DNA plasmid of TH2400CAT was used in which rat tyrosine hydroxylase gene was inserted into chloramphenicol acetyltransferase reporter gent. The transfection efficiency was 25-30% and the method was simple and reproducible. So, the method will be a good tool for transient transfection analysis.

• International Journal of Oral Biology
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• 제39권4호
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• pp.177-185
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• 2014

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the $Neon^{TM}$ and $NEPA21^{TM}$ electroporators were tested. $Neon^{TM}$ electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and $NEPA21^{TM}$ electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by $Neon^{TM}$ electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

• KSBB Journal
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• 제14권3호
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• pp.377-379
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• 1999

CHO/dhfr- 세포에 대해 LipofectAmine$^{TM}$을 이용한 유전자 이입 효율을 증가시키기 위하여 지질과 DNA의 최적 농도를 구하였다. Reporter 유전자로서 GFP 유전자를 이용하였으며, 여러 농도의 지질 DNA로 유전자 이입된 각 세포군에서 나타나는 green fluorescence intensity를 FACS 분석함으로써 유전자 이입 효율을 정량화 할 수 있었다. 그 결과 24-well plate에서 $2.0{\mu}L$LipofectAmine$^{TM}$과 $0.4{\mu}g$ DNA를 조합하여 사용했을 때 최적의 유전자 이입 효율이 나타남을 알 수 있었다. 또한, GFP는 유전자 이입 최적화를 수행하는 데에 여러가지 면에서 유용한 수단이 될 수 있음을 확인할 수 있었다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.113-117
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• 2009

Animals produced by somatic cell nuclear transfer (SCNT) using genetically modified cells are almost always transgenic, implying that this method is more efficient than the traditional pronuclear microinjection method. Most somatic cells for SCNT in animals are fetus-derived primary cells and successful gene integration in somatic cells will depend on transfection condition. The objective of this study is to evaluate the efficiency of electroporation (Microporator) and liposome reagents (F-6, F-HD, W-EX, W-Q, W-M) for tissue-type plasminogen activator (tPA) gene transfection and to estimate the overall efficiency of transfection of Korean native pig fetal fibroblast cells (KNPFF). Electroporation showed significantly higher transfection efficiency than liposome reagents with regard to the transfection of in vitro cultures in the early stages of development (41.7% with Microporator vs. 18.3% with F-6, 20.0% with F-HD 18.5% with W-EX, 5.0% with W-M and 6.3% W-Q,). Colonies identified as tPA-positives were treated once more with G418 for 10 to 14 days and growing colonies were selected again. When the cells of newly selected colonies were subjected to single-cell PCR, reselection of colonies following second round of G418 selection increased the rate of transgene integration per each colony. These results suggest that transfection with electroporation is the most efficient and the second rounds of G418 selection may be an effective method for transfection of porcine fetal fibroblast cells.

• 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.

• Bulletin of the Korean Chemical Society
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• 제30권4호
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• pp.927-930
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• 2009

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.426.3-427
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• 2002

The aim of this study was to enhance the transfection efficiency of emulsion-mediated gene expression by using chitosan, Conventional DNA/emulsion complexes and precondensed DNA/emulsion complexes were prepared by adding either naked or precondensed plasmids to cationic emulsion. The zeta potential. TEM, and size of transfection complexes were measured. In vitro transfection efficiency for boty complexes was also studied by several methods: flow cytometer, expression analysis by confocal microscope, RT-PCR, and in addition. (omitted)

• 한국정보통신학회논문지
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• 제18권2호
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• pp.482-487
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• 2014

본 연구는 testosterone이 지방세포생성에 미치는 영향과 그것에 대한 분자생물학적 조절기전을 역전사-중합효소 연쇄반응과 일시적인 형질전환 분석을 통해 조사하였다. 정소절제수술 마우스(CAST)에 비해 testosterone이 처리된 정소절제수술 마우스(CAST+T)는 백색지방조직 무게와 지방세포 특이유전자($PPAR{\gamma}$와 aP2)의 발현이 감소되었다. In vivo 결과와 일치되게, testosterone 처리는 분화된 3T3-L1세포에서의 triglyceride축적과 지방세포 특이유전자($PPAR{\gamma}$와 aP2)의 발현을 억제시켰다. Testosterone에 의해 활성화된 androgen receptor(AR)는 $PPAR{\gamma}$ transfection에 의해 유도된 luciferase reporter gene 활성을 억제하였다. 따라서 본 연구결과는 testosterone이 AR을 통해 지방세포분화에 대한 $PPAR{\gamma}$의 작용을 억제 조절한다는 것을 시사하고 있다.

• 한국양식학회지
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• 제13권1호
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• pp.29-37
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• 2000

In previously study we isolated several fish matrix attachment regions (MARs) capable of replicating the plasmid by itself. In this study we construct a fish expression vector pBaEGFP(+) containing mud loach ${\beta}$-actin promoter EGFP as reporter gene and SV40 signal. To analyze the effects of the fish expression vector respectively. The fish ARS containing constructs pBaEGFP(+)-ARSs were transfected cells with pBaEGFP(+)-ARS101 and pBaEGFP(+)-ARS223 reduced 10 days to 25 days and then was constant to 30 days after transfection while that of the control vector without ARS element was basal level. The intensity of both constructs showed about 30fold of the intensity compared with the control vector on 30days after transfection individually .E. coli back-transformation analysis shows that pBaEGFP(+)-ARS223 and pBaEGFP(+)-ARS905 maintain in episomal state at least 30 days after transfection. The result indicates that both may be able to replicate the vector in BF-2 cell. Therefore the matrix-attached ARSs enhancing expression of the reporter gene might be useful as a component o the expression vector for transgenic studies.

• 한국산학기술학회논문지
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• 제17권11호
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• pp.640-647
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• 2016

형광 간섭 현상을 최소화시켜 상대적으로 민감한 측정이 가능한 aequorin기반 luminescence기술은 $G_{{\alpha}16}$ 단백질 도입을 통해 세포 내부의 칼슘 이동 신호를 감지하여 G 단백질 결합 수용체(G protein-coupled receptor, GPCR)의 기능 분석을 가능하게 하는 세포 기반 분석 기술로 수용체 및 G 단백질 유전자 전달의 최적화 과정이 필수적이다. 본 연구를 위해 corticotropin releasing factor receptor subtype 2(CRF2) 수용체를 모델 시스템으로 CRF2와 $G_{{\alpha}16}$ 단백질이 구축된 세 가지 안정화 세포주를 제작하였고, 이들을 이용한 서로 다른 세 가지 조건의 임시 발현 세포주에서 작용제(sauvagine)와 길항제(K41498)의 반응성을 분석하여 최적의 유전자 전달 방법을 도출하고자 하였다. 그 결과 sauvagine 및 K41498의 농도에 따른 반응에서 CRF2-$G_{{\alpha}16}$ 안정화 세포주가 임시 발현 세포주보다 10배 이상의 유효신호 비율을 나타내었고(z'=0.77) 임시 발현 세포주의 경우 $G_{{\alpha}16}$의 안정화 발현 이후에 CRF2를 전달하는 경우가 다른 임시 발현 조건보다 2배 이상 높은 효율을 보였다(z'=0.84). 따라서 임시 유전자 전달 기술을 GPCR 세포 기능 분석 시스템에 활용할 경우 $G_{{\alpha}16}$ 단백질에 대한 안정화 세포주를 우선적으로 구축하고, 목표하는 다양한 수용체들을 단계적으로 발현시키는 것이 최선의 방법이라 판단된다.