• Journal of Pharmaceutical Investigation
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• v.34 no.4
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• pp.263-267
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• 2004

Branched and linear polyethylenimines (PEIs) have been studied as efficient and versatile agents for gene delivery in vitro and in vivo. PEIs exist in a linear or branched topology and are available in a wide range of molecular weight (Mw). Most studies have been done using PEIs with Mw higher than 10Kd. This study was aimed to test the transfection efficiency and the cell viability following gene delivery using PEI of Mw 2Kd, a relatively lower Mw cationic polymer. We used murine interleukin-2(mIL-2) plasmid DNA complexed with branched PEI 2Kd or 25Kd, and transfected them into a myoblast muscle cell line, C2C12. The cellular uptake of mIL-2 plasmid DNA was determined using quantitative polymerase chain reaction. RNA transcript levels were studied in the myoblast cells. Our results show that PEI 2Kd was as effective as PEI 25Kd in celluar gene delivery and transfection efficiency in C2C12 cells. Moreover, MTT assay indicated that PEI 2Kd/DNA complexes did not significantly reduce the cell viability regardless of N/P ratios. These results suggest that PEI of Mw 2Kd might play a role as effective and low toxic nonviral vector systems for muscular cell lines.

• Polymer(Korea)
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• v.31 no.5
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• pp.454-459
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• 2007

Gene therapy using low molecular weight water soluble chitosan (LMWSC) as polycationic polymer shows good biocompatibility, but low transfection efficiency. The mechanism of folic acid (FA) uptake in the cells to promote targeting and internalization could improve transfection rates. The objective of this study was to synthesize and characterize the WSCFA-DNA complex and evaluate their cytotoxicity, in vitro. In $^1H-NMR$ spectra, specific peaks appeared both of FA and LMWSC in $D_2O$. WSCFA nanoparticles have spherical shapes with particle size show below 110 nm. In the cell cytotoxicity test, the WSCFA-DNA complex showed high cell viability, in vitro. Gel electrophoresis showed condensed DNA within the carriers. hi vitro transfection efficiency was assayed by fluorescence spectroscopy WSCFA nanoparticles have less cytotoxicity, good DNA condensation and particle size around 110 nm, which makes them a promising candidate as a non-viral gene vector.

• Biomedical Science Letters
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• v.25 no.4
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• pp.398-406
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• 2019

Cancer immunotherapy using gene-modified tumor cells is safe and customized cancer treatment method. In this study, we made gene-modified tumor cells by transferring costimulatory molecules, 4-1BBL and OX40L, into tumor cells using lentivirus vector, and identified anti-cancer effect of gene-modified tumor cells in CT26 mouse colorectal tumor model. We construct pLVX-puro-4-1BBL, -OX40L vector for lentivirus production and optimized the transfection efficiency and transduction efficiency. The transfection efficiency is maximal at DNA:cationic polymer = 1:0.5 and DNA 2 ㎍ for lentivirus production. Then, the lentiviral including 4-1BBL and OX40L was used to deliver CT26 mouse tumor cells to establish optimal delivery conditions according to the amount of virus. The transduction efficiency is maximal at 500 μL volume of lentiviral stock without change in cell shape or growth rate. CT26-4-1BBL, CT26-OX40L significantly inhibited the tumor growth compare with CT26-WT or CT26-β-gal cell line. These data showed the possibility the use of genetically modified tumor cells with costimulatory molecule as cancer immunotherapy agent.

• Progress in Superconductivity and Cryogenics
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• v.19 no.1
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• pp.9-12
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• 2017

Recently, DNA vaccination is attracting attentions as a new therapeutic method for lifestyle diseases and autoimmune diseases. However, its clinical applications are limited because a safe and efficient gene transfer method has not been established yet. In this study, a new method of gene transfer was proposed which utilizes the jet injection and the magnetic transfection. The jet injection is a method to inject medical liquid by momentary high pressure without needle. The injected liquid diffuses in the bio tissue and the endocytosis is considered to be improved by the diffusion. The magnetic transfection is a method to deliver the conjugates of plasmid DNA and magnetic particles to the desired site by external magnetic field. It is expected that jet injection of the conjugates causes slight membrane disruptions and the traction of the conjugates by magnetic field induces the efficient gene transfer. In conclusion, the possibility of improvement of the gene expression by the combination of jet injection and magnetic transfection was confirmed.

• Bulletin of the Korean Chemical Society
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• v.22 no.1
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• pp.46-52
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• 2001

To evaluate the non-ionic polymer, poly(ethylene glycol) (PEG), as a component in cationic copolymers for non-viral gene delivery systems, PEG was coupled to polyethylenimine (PEI). We present the effects of different degrees and shapes of pegylation of PEI on cytotoxicity, water solubility and transfection efficiency. This work reports the synthesis and characterization of a series of cationic copolymers on the basis of the conjugates of PEI with PEG. The modified molecules were significantly less toxic than the original polymer. Moreover, the chemical modification led to enhancement of their solubility. The comparison of pegylated PEIs with different degrees of derivation showed that all the polymers tested reached comparable levels of transgene expression to that of native PEI. As assessed by agarose gel electrophoresis, even highly substituted PEI derivatives were still able to form polyionic complexes with DNA. However, aside from an increase in solubility and retention of the ability to condense DNA, methoxy-PEG-modified PEIs resulted in a significant decrease in the transfection activity of the DNA complexes. In fact, the efficiency of the copolymer was compromised even at a low degree of modification suggesting that the PEG action resulting from its shape is important for efficient gene transfer. The mode of PEG grafting and the degree of modification influenced the transfection efficiency of PEI.

• KSBB Journal
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• v.16 no.6
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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.

• Polymer(Korea)
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• v.29 no.1
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• pp.69-73
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• 2005

To overcome the main disadvantages of non-viral gene delivery systems such as repeated administration due to the low transfection efficiency, poly(D,L-lactide-co-glycolide) was applied to encapsulate pDNA in its microsphere formulation. Free pDNA or various ratios (w/w) of chitosan/pDNA complexes was used for encapsulation, with the resulting encapsulation efficiency of 44%, 5%, and 8% for free pDNA, 0.7:1 and 1:1 ratios, respectively. Scanning electron micrographs of poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres encapsulating pDNA or chitosan-condensed pDNA revealed a smooth spherical shape immediately after microsphere preparation and a collapsed porous shape in 41 days due to the degradation of PLGA. In vitro release profile showed that the 0.7:1 (w/w) ratio formulation exerted 47% release in 26 days, whereas free pDNA or 1:1 (w/w) ratio formulation did only 15% or 32%, respectively.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.4
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• pp.581-589
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• 1993

A plasmid vector, $RSVLTR/{\beta}G2$, containing lacZ gene under the control of the RSVLTR promoter were transfected into chicken embryo fibroblasts by three different transfection methods. Calcium phosphate, lipsome and DEAE-dextran techniques were applied for transfection of chicken cells. A histochemical assay with X-gal was used as a simple method for screening transfected cells. Plasmid $RSVLTR/{\beta}G2$ was expressed proficiently in the chicken embryo fibroblast. Calcium phosphate-DNA precipitate transfection resulted in the highest efficiency for transient expression of $RSVLTR/{\beta}G2$. Transfected cells formed colonies on the 9th day of incubation indicating stable transformation of the inserted plasmid.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.451-454
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• 2005

Introduction of foreign genes into brain cells, such as neurons and astrocytes, is a powerful approach to study the gene function and regulation in the neuroscience field. Calcium phosphate precipitates have been shown to cause cytotoxicity in some mammalian cells and brain cells, thus leading to low transfection efficiency. Here, we describe a retrovirus-mediated gene delivery method to transduce foreign genes into brain cells. In an attempt to achieve higher gene delivery efficiency in these cells, we made several changes to the original method, including (1) use of a new packaging cell line, Phoenix ampho cells, (2) transfection of pMX retroviral DNA, (3) inclusion of 25 mM chloroquine in the transduction, and (4) 3- 5 h incubation of retroviruses with target cells. The results showed that the modified protocol resulted in a range of 40- 60% gene delivery efficiency in neurons and astrocytes. Furthermore, these results suggest the potential of the retrovirus-mediated gene delivery protocol being modified and adapted for other transfection-refractory cell lines and primary cells.