• Bulletin of the Korean Chemical Society
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• v.26 no.8
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• pp.1209-1213
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• 2005

Nuclear membrane is one of the main barriers in intracellular delivery of genetic materials. The previous report showed that glucocorticoid receptor dilated the nuclear pore to 60 nm in the presence of a ligand. It was also suggested that the transport of genetic material to nucleus might be facilitated by glucocorticoid. In this study, the effect of glucocorticoid preincubation in the polymeric gene delivery was investigated. The cells were preincubated with dexamethasone, a potent glucocorticoid, and transfection assays were performed with polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer. As a result, the transfection efficiency of PEI or PAMAM to the cells in the presence of dexamethasone was enhanced, compared to the cells without dexamethasone. This effect was not observed in the cells preincubated with cholesterol. The polymer/DNA complex was stable in the presence of dexamethasone. In addition, the cytotoxicities of the polymeric carriers to the cells were observed in the presence of dexamethasone. In conclusion, dexamethasone enhances the transfection efficiency of polymeric carriers and may be useful in the development of polymeric gene carriers.

• Korean Chemical Engineering Research
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• v.43 no.2
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• pp.313-317
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• 2005

Synthetic gene carriers such as poly-cationic polymers easily form complexes with plasmid DNA which contains negative charge. Chitosan is a polysaccharide that demonstrates much potential as a gene delivery system. The ability of depolymerized chitosan to condense DNA was determined using electrophoresis. Dynamic laser scattering and scanning electron microscopy were used to examine the size and the morphology of the chitosan/DNA complex. Parameters such as chitosan molecular weight and charge density influenced the complex size and the DNA amount condensed with chitosan. The cell viabilities in the presence of chitosan ranged between 84-108% of the control in all experiments. Gene expression efficacy using chitosan/DNA complex was enhanced in 293 cells relative to that using naked DNA, although it was lower than that using lipofecamine. Transfection efficacy using low molecular weight chitosan (Mw=8,517) was higher than those of the control and the other chitosan (MW=4,078). The low molecular weight chitosan (MW=8,517) with a high charge density (18.32 mV) fulfilled the requirements for a suitable model gene delivery system with respect to the condensing ability of DNA, complex formation, and transfection efficacy.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.189-194
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• 2006

Sperm-mediated gene transfer(SMGT) can be used to transfer exogenous DNA into the oocyte at fertilization. The main objective of this study was to assess efficiency of transferring mitochondrial DNA(mtDNA) fragment into boar spermatozoa in either presence or absence of liposome and quality of transfected spermatozoa. The mtDNA of chicken liver was isolated and purified by phenol and alkaline lysis extraction, and it was inserted to plasmid. The genome of transfected spermatozoa treated with DNase I was purified by alkaline lysis, and then amplified by the PCR analysis. After electrophoresis, DNA quantitation of each well was calculated by comparison of the band intensity with standard. As a result, exogenous DNA was composed of mtDNA fragment(1.2 kb) and plasmid(2.7 kb). On the other hand, efficiency of transfection by liposome($9.0{\pm}0.34ng/{\mu}l$) in SMGT was higher than that by DNA solution($6.9{\pm}0.53ng/{\mu}l$). However, there was no significant difference. Transfering exogenous DNA into spermatozoa was completed within 90 min of incubation. In another experiment, there were significant (p<0.05) differences between transfected spermatozoa using both DNA solution and DNA/liposome completes with unheated spermatozoa for viability ($70.8{\pm}1.80$ and $68.0{\pm}2.16%$ vs. $83.3{\pm}1.69%$, respectively) and motility($78.7{\pm}1.59$ and $79.3{\pm}2.14%$ vs. $86.7{\pm}1.59%$, respectively). This study indicates that exogenous mtDNA can be efficiently transferred into boar spermatozoa regardless of the presence of liposome, and transfected spermatozoa can also use insemination and in vitro fertilization to generate transgenic pig.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.351.1-351
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• 1995

No Abstract, See Full Text

• Journal of Life Science
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• v.24 no.12
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• pp.1269-1275
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• 2014

TALEN is a newly developed gene engineering method to knock out specific genes. It contains a DNA binding domain and a Fok1 nuclease domain in the TALEN plasmid. Therefore, the engineered TALEN construct can bind to any region of genomic DNA and cut the target nucleotide, thereby inducing mutation. In this study, we constructed two TALEN constructs targeted to a protein initiation codon (DBEX2) or the 25th upstream region (DBPR25) to enable mRNA synthesis of SETDB1 HMTase. We performed the TALEN cloning in two steps. The first step was from module vectors to pFUS array vectors. We confirmed successful cloning with a colony PCR experiment and Esp31 restriction enzyme digestion, which resulted in a smear band and a 1 Kb insert band, respectively The second step of the cloning was from a pFUS array vector to a mammalian TALEN expression vector. The engineered TALEN construct was sequenced with specific primers in an expression vector. As expected, a specific array from the module vectors was shown in the sequencing analysis. The specific module sequences were regularly arrayed in every 100 bp, and SETDB1 expression totally disappeared in the TALEN-DBEX2 transfection. PCR amplification targeting of DBEX2 was performed, and the PCR product was digested with a T7E1 restriction enzyme. The expression of SETDB1 was down-regulated in the TALEN-DBPR25 transfection. Morphological changes were also observed in the two TALEN constructs with transfected HeLa cells. These results suggest that the engineered TALEN constructs in two strategic approaches are very useful to knock-out of the SETDB1 gene and to study gene function.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.81-87
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• 2008

Production of transgenic animals for studying specific gene has been limited due to a low efficiency, lack of skilled researchers and the need for expensive equipment. Currently, the boar spermatozoa as a vector to deliver exogenous DNA into the oocyte were used to improve the efficiency of transfection rate. In this study, we revealed that the optimal conditions for DNA uptake in spermatozoa by liposome were to 90 min of incubation, $17^{\circ}C$, $10^5$ spermatozoa, 4 ng/ml of exogenous DNA and 0.5% (v/v) liposome, without damage to fertility. In addition, the developmental rate to the blastocyst stage of embryo in control group was significantly higher than those embryos with exogenous DNA and liposome, whereas there were no significant differences in embryo development between the liposome and type of DNA. The transfection rates of embryo using treated spermatozoa with both liposome and circular DNA were higher than those using linear DNA. These findings raise the possibility thattreated spermatozoa with liposome/DNA complexes could be used in in vitro fertilization, and the exogenous DNA transferred into the oocytes. Taken together, we demonstrated that liposome a vector for the uptake of exogenous DNA in boar spermatozoa could improve the efficiency of sperm-mediated gene transfer in creating transgenic pig and the other domestic transgenic animals.

• Macromolecular Research
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• v.14 no.3
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• pp.348-353
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• 2006

For enhancing the gene delivery efficiency of polyplexes, a new formulation was developed using PEI conjugated Pluronic F127 copolymer as an effective additive. Low molecular weight, branched polyethylenimine Mw 600 (LMW BPEI 600) was conjugated to the terminal end of Pluronic F127. The PEI-modified Pluronic copolymers formed a micellar structure in aqueous solution, similar to that of unmodified Pluronic copolymer. PEI modification of Pluronic copolymer increased the size of micelles while concomitantly raising the critical micelle concentration (CMC). The PEI-modified Pluronic copolymer was used as a micellar additive to enhance the gene transfection efficiency of pre-formulated polyelectrolyte complex nanoparticles composed of luciferase plasmid DNA and branched PEI Mw 25k (BPEI 25k) or polylysine Mw 39k (PLL 39k). The luciferase gene expression levels were significantly enhanced by the addition of the BPEI-modified Pluronic copolymer for the two formulations of BPEl and PLL polyplexes. The results indicated that the BPEI-modified Pluronic copolymer micelles ionically interacted on the surface of DNA/BPEI (PLL) polyplexes which might facilitate cellular uptake process.

• Biomedical Science Letters
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• v.15 no.4
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• pp.265-272
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• 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.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.198-203
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• 2007

DNA transfection is a powerful tool for studying gene functions. The $Ca^{2+}$-phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of $Ca^{2+}$-phosphate precipitation under microscope. Cerebral glial cells were grown until ${\sim}70-80%$ confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and 319 hippocampal neurons were plated onto the glial layer Formation of fine $DNA/Ca^{2+}$-phosphate precipitates was induced using Clontech $CalPhos^{TM}$ Mammalian Transfection Kit, and the size ($0.5-1\;{\mu}m$ in diameter) and density(about 10 particles/$100\;{\mu}m^2$) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of $pEGFP-CaMKII{\alpha}$, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.279-283
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• 2001

Cationic lipid emulsion system consisting of 1, 2-dioleoyl-sn-slycero-3-trimethyl-ammonium-propane(DOTAP) and plasmin DNA with various counterions in the lipid headgroups were prepared. The transfection activity of the cationic lipid emulsion systems was then investigated in vitro and in vivo. The complex formation of plasmid DNA lipid emulsion was affected by the counterions through charged headgroup repulsion and also by the salt concen-tration in the media. As such , the transfection activity of the DOTAP emulsion system can be controlled by changing the counterions.