• Polymer(Korea)
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• v.31 no.6
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• pp.555-561
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• 2007

To obtain low molecular weight water soluble chitosan (LMWSC) with various molecular weights, chitosan oligosaccharides (COS) with lactic acid was separated by using ultrafilteration technique and LMWSC with a free amine group was prepared by the novel salts-removal method. The characterization of LMWSC removed the lactic acid and degree of deacetylation (DDA) were identified by FT-IR and $^1H-NMR$ spectra. Polydispersity index (PDI) was $1.278{\sim}1.499$, which indicates a relatively molecular weight distribution. To identify the potential as a gene carrier, we confirmed the transfection efficiency of COS fractioned according to molecular weight successfully and the salt-removed LMWSC using 293T cell. Also, LMWSC derivatives prepared for improvement transfection efficiency were evaluated using Balb/C mice.

• 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.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.55-55
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• 2003

The random insertion of useful gene in genome has been a common method to produce transgenic animals. This method is inefficient for induction of high levels gene expression in transgenic animals. To improve this limit, we tried to develop the system which target the gene at the specific genomic region. Thus, in our experiment, the vector system to target the human thrombopoietin (TPO) gene was developed. Targeting vector including TPO, neo and DT genes was transfrcted into bovine embryonic fibroblasts (bEF) or bovine ear skin fibroblasts (bESF). First of all, we determined concentration of the geneticin (G418) for selection of transfected cell lines. Our results showed that 1200 and 900 $\mu\textrm{g}$/ml of G418 were the most proper for selection of transfscted bEF and bESF cells. In this study, lipofectamine was used as a transfection reagent. Thus, the proper ratio of DNA:lipofectamine for transfection was also required to elevate targeting efficiency in primary mammalian cells. Our result indicates that the most proper ratios of DNA:lipofectamine were 4:2 and 1:2 in bEF and bESF cells. According to the optimized these conditions, single colonies were picked following transfection and were analyzed by PCR. More than 90% of the single colonies have TPO gene. However, there were no colonies with targeted TPO at the specific genomic region. Therefore, further experiments to select the specifically targeted colonies and to find more efficient methods such as reducing selection time and shortening a size of TPO gene are required.

• Bulletin of the Korean Chemical Society
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• v.32 no.2
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• pp.651-655
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• 2011

We synthesized a novel cationic dendrimer consisting of a poly(amidoamine) dendrimer (PAMAM, generation 4) backbone with both L-arginine (Arg) at the termini and 6-aminohexanoic acid (Ahx) between the original core polymer and the peripheral Arg units. The sequential chemical modification of PAMAM G4 with Ahx and Arg resulted in higher transfection efficiency with much less cytotoxicity. PAMAM G4-Ahx-Arg formed stable polyplexes at weight ratios of 8:1 or higher (polymer: plasmid DNA), and the mean polyplex diameter was $180{\pm}20nm$. PAMAM G4-Ahx-Arg showed much higher transfection ability than PAMAM G4 or PAMAM G4-Ahx. Furthermore, PAMAM G4-Ahx-Arg was much less cytotoxic than PEI25KD and PAMAM G4-Arg. In addition to Arg grafting of the PAMAM dendrimer, which endows a higher transfection capability, the addition of Ahx spacer increased dendrimer hydrophobicity, introduced flexibility into the conjugated amino acids, and reduced cytotoxicity. Overall, it appears that the concomitant modification of PAMAM with Ahx and Arg could lead to new PAMAM conjugates with better performances.

• Archives of Pharmacal Research
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• v.24 no.1
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• pp.1-15
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• 2001

Gene therapy has emerged as a new concept of therapeutic strategies to treat diseases which do not respond to the conventional therapies. The principle of gene therapy is to Introduce genetic materials into patient cells to produce therapeutic proteins in these cells. Gene therapy is now at the stage where a number of clinical trials have been carried out to patients with gene-deficiency disease or cancer. Genetic materials for gene therapy are generally composed of gene expression system and gene delivery system. For the clinical application of gene therapy in a way which conventional drugs are used, researches have been focused on the design of gene delivery system which can offer high transfection efficiency with minimal toxicity. Currently, viral delivery systems generally provide higher transfection efficiency compared with non-viral delivery systems while non-viral delivery systems are less toxic, less immunogenic and manufacturable in large scale compared with viral systems. Recently, novel strategies towards the design of new non-viral delivery system, combination of viral and non-viral delivery systems and targeted delivery system have been extensively studied. The continued effort in this area will lead us to develop gene medicine as "gene as a drug" in the near future.

• Journal of Pharmaceutical Investigation
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• v.30 no.1
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• pp.1-12
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• 2000

Gene therapy is a method for the treatment of diseases with introducing the gene-engineered materials into a patient with gene-deficiency disease (e.g. cystic fibrosis) or cancer to produce a therapeutic protein in a patient's cells. Successful gene therapy requires establishing both gene expression systems and delivery systems. Viral and non-viral vectors have been used for gene delivery. Viral vectors have a high transfection efficiency, but are limited in relations to issues of safety, toxicity and immunogenecity. Non-viral vectors are easy to prepare and relatively safe. However, non-viral vectors have a low transfection efficiency. Cationic liposomes are the most available among non-viral vectors. Cationic liposomes have been used to transfect cells both in vitro and in vivo experiments. Besides, several formulations containing cationic lipid are being used in clinical trials in cases of cystic fibrosis or cancer. A crucial subject to the further development of gene delivery vectors will be a long-term gene expression with following characteristics; protecting and deliverying DNA efficiently, non-toxic and non-immunogenic, and easy to produce in large scale.

• Bulletin of the Korean Chemical Society
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• v.29 no.3
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• pp.666-668
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• 2008

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2045-2049
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• 2012

Objective: To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms. Methods: Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviral vector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cells without transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) served as control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR and Western blot assays were employed to detect the inference efficiency. The morphology of cells was observed under a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays were conducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). After treatment with $COCl_2$ for 24 h, the mRNA and protein expression of hypoxia inducible factor -$1{\alpha}$ (HIF-$1{\alpha}$) was determined. Results: Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfully constructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interference efficiency was $79.2{\pm}0.026%$. At 48 h after transfection, the Daudi cells became shrunken, had irregular edges and presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 with prolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-$1{\alpha}$ remained unchanged in three groups (P>0.05) but the protein expression of HIF-$1{\alpha}$ markedly increased (P<0.05). However, in the sh-SENP1-Daudi group, the protein expression of HIF-$1{\alpha}$ remained unchanged Conclusion: SENP1-shRNA can efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. These findings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.2
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• pp.158-164
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• 2005

A practical approach was proposed to produce transgenic chimeric chickens using blastodermal cells (BCs). The chicken BCs were mechanically dissociated and transferred into the recipient eggs that had been exposed to 500 rads irradiation of$^{60}Co$ and windowed on the equatorial plane. Chimeric chickens were generated using two models: the crosses (MXL) from Black Minors (ii,EE,b/b) ♂${\times}$Barred Leghorns (ii,ee,B/-) ♀ as donors and White Leghorns (WL, II) as acceptors (Model 1), or the Black Heifengs (BH, ii,EE,bb) as donors and Hua-xing white (HW, II) as recipients (Model 2). The treated eggs were incubated in their original shells in normal conditions until hatching. Green fluorescent protein (GFP) gene was transferred into the BCs derived from MXL and BH via lipofectamine and the pEGFP-C1, and transfection efficiency into the BCs was examined under a fluorescent microscope. Potential transgenic chimeras were selected based on the proposed methods in this study. Using the fresh BCs, the best rate of phenotypic chimeras was 6.7% and 26.0% in model-1 groups, and model-2 groups, respectively. We also described the optimized conditions for transfection. Although 30% of the BCs transfected in vitro emitted green light under an inverted fluorescent microscope, no embryos injected with the transfected BCs expressed foreign GFP gene at 3-4 days.