• 폴리머
• /
• 제31권5호
• /
• pp.454-459
• /
• 2007

본 연구에서는 folic acid(FA)가 복합화된 저분자량 수용성 키토산(LMWSC) 나노입자(water soluble chitosan-folic acid nanoparticle, WSCFA)를 제조하고, 또한 DNA와 나노복합체 합성 및 특성을 분석함으로써 in vitro에서 세포내 독성을 평가하였다. WSCFA 합성을 확인하기 위하여 분광학적 분석 방법을 사용하여 분석하였으며, WSCFA 나노입자는 110 nm 이하의 입자 크기인 구형의 형태를 가지고 있음을 알 수 있었다. In vitro 세포내 독성 실험에서, WSCFA-DNA 복합체는 세포내 독성을 전혀 나타내지 않음으로 높은 세포 생존율을 보여주었다. 전기영동 실험을 통해 WSCFA의 DNA 응축능력을 확인하였고, in vitro에서의 전이효율은 형광 광도계에 의해 평가하였다.

• 대한의생명과학회지
• /
• 제10권3호
• /
• pp.187-194
• /
• 2004

A short peptide corresponding to the nuclear localization signal (NLS) of human immunodeficiency virus (HIV)-l Tat protein, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, was employed to improve the efficiency of cellular uptake of nucleic acids. The peptide was first mixed with a reporter plasmid and then with cationic liposomes to form a tripartite complex of DNA/peptide/liposomes (DPL). Transfection efficiency of the DPL complex was compared with that of the conventional DNA/liposomes (DL) complex. When the DPL complex was formed with various cationic liposomes, DOTAP/DOPE (DP) liposome exhibited superior transfection efficiency to other liposomes tested in vitro. With the inclusion of the peptide, the DPL complex showed much enhanced transfection in various cancer cell lines. Particularly, transfection of the DPL complex in serum increased cellular uptake of a transgene up to 2 fold when compared with that in a serum free condition. Further, when the DPL complex was infused through the ureteric route of a rat, transfection efficiency was shown to be better in reporter gene expression than that obtained with the DL complex. This study shows that the DPL complex that is easy to formulate can be employed for much enhanced cellular uptake of a trans gene.

• Journal of Pharmaceutical Investigation
• /
• 제28권2호
• /
• pp.93-98
• /
• 1998

We have developed liposomes which can be easily prepared with inexpensive lipid, have enhanced stability, and can efficiently deliver DNA into the COS-l cells, Liposome formulations were prepared using cationic materials such as dimethyldioctadecyl ammonium bromide (DDAB), cetyltrimethyl ammonium bromide(CTAB), We investigated the effect of cationic liposome formulations on in vitro DNA transfection, DDAB-containing liposomes showed increased transfection efficiency which was 3.2-fold as much as that by $Lipofectin^{\circledR}$, but CTAB-containing liposomes were inactive in gene transfection. The effect of colipid of DDAB-containing liposome was also investegated. As a colipid, dioleylphosphatidylethanolamine(DOPE) and cholesterol did altered the transfection efficiency of DDAB-containing liposomes. And increased DDAB concentration lowered the transfection efficiency. The optimum amount of liposomal formulation was $10\;{\mu}M$ for $1\;{\mu}g$ of DNA. In the experiment of stability, DOPE-containing liposomes formulation showed a broad size distribution and separation of two major peaks on a 5th day of preparation, but liposomes containing cholesterol was stable for 10 days. DDAB-containing liposomal DNA delivery system was prepared easily and was stable.

• BMB Reports
• /
• 제33권6호
• /
• pp.476-482
• /
• 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).

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

• 대한의생명과학회지
• /
• 제15권4호
• /
• pp.265-272
• /
• 2009

Recently, mesenchymal stem cells (MSCs) began to be utilized as a vehicle for ex vivo gene therapy based on their plasticity. Effective and safe transfection of therapeutic genes is a critical step for genetic modification of MSCs. Therefore, optimization of in vitro gene delivery into MSCs is essential to provide genetically modified stem cells. In this study, various cationic liposomes, O,O'-dimyristyl-N-lysyl aspartate (DMKD), DMKD/cholesterol, O,O'-dimyristyl-N-lysyl glutamate (DMKE), DMKE/cholesterol, and N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl sulfate (DOTAP)/cholesterol, were mixed with plasmid DNA encoding luciferase (pAAV-CMV-Luc) at varied ratios, and then used for transfection to MSCs under varied conditions. The MSCs were also transfected by electroporation under varied conditions, such as voltage, pulse length, and pulse interval. According to the experimental results, electroporation-mediated transfection was more efficient than cationic liposome-mediated transfection. The best MSC transfection was induced by electroporation 3 times pulses for 2 ms at 200 V with 10 seconds of a pulse interval.

• 생명과학회지
• /
• 제17권2호통권82호
• /
• pp.198-203
• /
• 2007

형질전환은 유전자의 기능을 이해하는데 매우 중요한 기법이다. $Ca^{2+}$-인산 침전법은 시간과 비용이 저렴하여 가장 흔히 사용된다. 그러나 성숙 신경세포는 어린 신경세포나 다른 세포종에 비하여 형질전환이 어렵고 쉽게 죽는다. 본 연구에서는 Clontech사의 $CalPhos^{TM}$ Mammalian Transfection 방법을 수정하여 성숙한 신경세포를 효율적으로 형질전환할 수 있는 방법을 고안하였다. 대뇌 신경교세포를 DMEM/10% 말혈청에서 70-80% confluence까지 키우고 배지를 혈청이 첨가되지 않은 Neurobasal/Ara-C로 바꾸어 주어 더 이상 신경교세포가 분열하지 않게 한 다음, 여기에 E19 해마신경세포를 접종하여 배양하였다. $DNA/Ca^{2+}$-인산 침전물은 Clontech사의 $CalPhos^{TM}$ Mammalian Transfection Kit을 이용하여 크기($0.5-1\;{\mu}m$ in diameter) 및 농도(약 10 particles/$100\;{\mu}m^2$)를 배지에서 배양시간을 변화시켜 적당히 조절하였다. 이렇게 하면 in vitro에서 2주 이상 배양한 신경세포도 24-well plate 한 well당 10-15개의 형질전환된 건강한 신경세포를 얻을 수 있었다. 이 방법의 효용성을 검증하기 위하여 연접단백질인 $EGFP-CaMKII{\alpha}$ 융합단백질과 RFP 단백질 유전자(각각 $pEGFP-CaMKII{\alpha}$ 및 pDsRed2)를 형질전환한 결과 전자는 점박이 모양, 후자는 세포전체에 퍼진 양상의 표현을 관찰할 수 있었다. 따라서 본 연구는 성숙한 신경세포를 효율적으로 형질전환할 수 있는 방법을 제공한다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제21권5호
• /
• pp.648-656
• /
• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Journal of Korean Neurosurgical Society
• /
• 제37권1호
• /
• pp.48-53
• /
• 2005

Objective: The purpose of this study is to elucidate in vitro responses to combined gamma knife irradiation and p53 gene transfection on human malignant glioma cell lines. Methods: Two malignant human glioma cell lines, U87MG (p53-wild type) and U373MG (p53-mutant) were transfected with an adenoviral vector containing p53 (MOI of 50) before and after applying 20Gy of gamma irradiation. Various assessments were performed, including, cell viability by MTT assay; apoptosis by annexin assay; and cell cycle by flow cytometry, for the seven groups: mock, p53 only, gamma knife (GK) only, GK after LacZ, LacZ after GK, GK after p53, p53 after GK. Results: Cell survival decreased especially, in the subgroup transfected with p53 after gamma irradiation. Apoptosis tended to increase in p53 transfected U373 MG after gamma irradiation (apoptotic rate, 38.9%). The G2-M phase cell cycle arrest markedly increased by transfecting with p53, 48 hours after gamma knife irradiation in U373 MG (G2-M phase, 90.8%). Conclusion: These results suggest that the in vitro effects of combined gamma knife irradiation and p53 gene transfection is an augmentation of apoptosis and G2-M phase cell cycle arrest, which are more exaggerated in U373 MG with p53 transfection after gamma knife irradiation.

• KSBB Journal
• /
• 제13권4호
• /
• pp.418-423
• /
• 1998

Cellular transfections with cationic liposomes are widely empolyed for gene and oligonucleotide transfer in vitro because of their safety and ease of use. However, they still suffer from the low transfection efficiency comparing with viral vectors. Substantial effort shave been focused on increasing transfection efficiency by supplementing the liposome/DNA complexes(lipoplex) with various components. In this work, we tired three kinds of supplements, Poly-L-lysine(PLL), transferrin and a mixture of anionic lipids(PS/PE/PC), to study their effects on gene transfer yield and gene expression efficiency. PLL, a polycationic polymer, enhanced gene transfer yield by 3 times but the gene expression efficiency was increased only by 1.5 times. this result implies that PLL can enhance the transfection efficiency mainly by increasing the rate of outermembrane transport of lipoplex into the cells. On the other hand, transferrin which can facilitate the gene transfer via ligand-receptor interaction gave not only increased gene transfer yield but also enhanced gen expression efficiency by 2.8 times. Transferrin seems to contribute to the escape of plasmid from endosomes through ligand-receptor recycle mechanism. When the cells were treated with a mixture of anionic lipids for 3 hours before the transfection, gene transfer yield was slightly decreased but the gene expression efficiency was enhanced by 1.9 times. This is presumably due to the accelerated liposome-plasmid dissociation by the anionic lipids, and the increased delivery of plasmid to the nucleus. According to these results, it is clear that the supplementation to ameliorate transfection efficiency with cationic liposomes should be contrived in the direction of increasing delivery of plasmid.