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

• Journal of The Korean Society of Inherited Metabolic disease
• /
• v.5 no.1
• /
• pp.9-17
• /
• 2005

Homocystinuria is a metabolic disorder caused by a deficiency of cystathionine ${\beta}$-synthase (CBS). Patients with homocystinuria show clinical symptoms such as mental retardation, lens dislocation, vascular disease with life-threatening thromboembolisms and skeletal deformities. Generally, the major treatments for CBS deficiency include pharmacologic doses of pyridoxine or dietary restriction of methionine. However, there is no effective treatment for this disease up till today and gene therapy can be an attractive novel approach to treatment of the disease. We investigated whether a recombinant adeno-associated virus could be used as a CBS gene transfer vector to reduce the excessive homocysteine level in the homocystinuria mouse model. Recombinant adeno-associated virus vector encoding the human CBS gene (rAAV-hCBS), driven by EF1-a promoter, was infused into CBS-deficient mice ($CBS^{-/-}$) via intramuscular (IM) and intraperitoneal (IP) injection. IP injection was more efficient than IM injection for prolongation of lives and reduction of plasma homocysteine levels. After 2 weeks of gene transfer by IP injection, serum homocysteine level was significantly decreased in treated mice compared with the age-matched controls and the life span was extended about 1.5 times. Also, increased expression of CBS gene was observed by immunohistochemical staining in livers of treated $CBS^{-/-}$ mice and microvesicular lipid droplets was decreased in cytoplasm of liver. These results demonstrate the possibility and efficacy of gene therapy by AAV gene transfer in homocystinuria mice.

• Journal of Aquaculture
• /
• v.10 no.4
• /
• pp.409-415
• /
• 1997

The carp $\beta$-actin regulatory sequences and RSV/LTR promoter were tested whether they are functinal to express linked structure gene (chloramphenicol acetyltransferas, CAT) in olive flounder (Paralichthys olivaceus) by determining the patterns of gene expression following intramuscular in vivo direct injection. The injection experiments with various concentrations of both pRSVCAT and pFV4CAT clearly revealed the effectiveness of DNA dosage on expression of CAT. The increase of CAT activity was linear in both plasmids, and maximal CAT activity was obtained with 100 ug of pFV4CAT injection. The amounts of CAT expression with pFV4CAT-injected fist were higher than those with pRSVCAT-injected fish. CAT activity was readily detectable as early as one day after injection, slightly increased at day 2, and declined over time. Most amount of DNA intramuscularly injected into olive flounder muscles persisted extrachromosomally without showing any integrated or replicated form in vivo.

• Molecules and Cells
• /
• v.39 no.10
• /
• pp.728-733
• /
• 2016

Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD.

• Journal of Aquaculture
• /
• v.12 no.1
• /
• pp.71-77
• /
• 1999

The potential utility of injection media (sucrose, PEG, and liposome) was demonstrated for direct gene transfer into olive flounder (Paralichthys olivaceus) muscles. Based on the use of sucrose (final cone. 20%), PEG 8,000 (final cone. 10%) or liposome (twice us of DNA injected), the present injection strategy significantly improved the level of transgene expression as well as persistent duration of expression. The increased amounts of expression in DNA injection with sucrose, PEG, and liposome were as high as from 2.1 to 4.9-folds of conventional TE-based DNA injection. The best result was obtained from injections of liposome-encapsulated DNA in which the expression was detectable at least 32 days after injection when compared to only 8-16 days from TE-based injections.

• Journal of Embryo Transfer
• /
• v.13 no.2
• /
• pp.97-106
• /
• 1998

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% $CO_2$ incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.98-98
• /
• 2003

Direct gene transfer to mammalian tissues has significant potential for gene therapy and transgenesis. Liposome-mediated in vivo transfection has begun to gain attention as an alternative to viral vectors, and may also be a good mode of transfection in gene transfer. Interestingly, polymerized cationic liposomes are reported to be very stable in the bloods and efficient for in vivo gene transfer. To examine a possible gene delivery in vivo, we investigated the efficacy and safety of the liposome-mediated gene transfer using vein injection in chick or mouse as model animals. The number of injected pGFP-LacZ using either a commercial or home-made liposomes was 8 and 19 at 16 and 7 day of hatch, respectively. One of injected chick of each experiments was analyzed and the rest is being bred. In mouse, 4/22 showed expression of pGFP-LacZ but 8/22 showed no expression and the remaining animals are also being bred. After injection of liposome/pGFP-LacZ complex into wing vein of 7 or 16 day-old chick, pGFP-LacZ was detected in various tissues isolated from not only young chick but also old chick were turned out to possess. exogenous DNA. Transcripts and proteins of the transgene were also detected by RT-PCR or histochemical analysis, respectively. These results suggest that injected DNA were inserted to genome and produced mRNA and proteins in various tissues and may give an important tools for effective gene delivery in gene therapy or transgenesis.

• Reproductive and Developmental Biology
• /
• v.32 no.1
• /
• pp.9-14
• /
• 2008

Efficient gene transfer into hematopoietic stem cells is a great tool for gene therapy of hematopoietic disease. Retrovirus have been extensively used for gene delivery and gene therapy. However, current in vitro gene transfer has some obstacles suck as induction of differentiation loss of self-renewal capacity, and down-regulation of homing efficiency for in vitro hematopoietic stem cells transplantation. To overcome these problems, we developed efficient in vitro retroviral transfer technique by direct intra-bone marrow injection (IBM). We identified effective retrovirus gene transfer in bone marrow hematopoietic cells in vitro. Two weeks after retrovirus transfer via IBM injection, we observed stable EGFP gene expression in bone marrow, lymph node, spleen, and liver cells. In addition, $6.4{\pm}2.7%$ of hematopoietic stem/progenitor cells were expressed EGFP transgene from flow cytometry analysis. Our results demonstrate that in vitro retrovirus gene transfer via IBM injection can provide a viable alternative to current or moo gene transfer approach.

• The Korean Journal of Nuclear Medicine
• /
• v.38 no.2
• /
• pp.152-160
• /
• 2004

Radioiodide uptake in thyroid follicular epithelial cells, mediated by a plasma membrane transporter, sodium iodide symporter (NIS), provides a first step mechanism for thyroid cancer detection by radioiodide injection and effective radioiodide treatment for patients with invasive, recurrent, and/or metastatic thyroid cancers after total thyroidectomy. NIS gene transfer to tumor cells may significantly and specifically enhance internal radioactive accumulation of tumors following radioiodide administration, and result in better tumor control. NIS gene transfers have been successfully performed in a variety of tumor animal models by either plasmid-mediated transfection or virus (adenovirus or retrovirus)-mediated gene delivery. These animal models include nude mice xenografted with human melanoma, glioma, breast cancer or prostate cancer, rats with subcutaneous thyroid tumor implantation, as well as the rat intracranial glioma model. In these animal models, non-invasive imaging of in vivo tumors by gamma camera scintigraphy after radioiodide or technetium injection has been performed successfully, suggesting that the NIS can serve as an imaging reporter gene for gene therapy trials. In addition, the tumor killing effects of I-131, ReO4-188 and At-211 after NIS gene transfer have been demonstrated in in vitro clonogenic assays and in vivo radioiodide therapy studies, suggesting that NIS gene can also serve as a therapeutic agent when combined with radioiodide injection. Better NIS-mediated imaging and tumor treatment by radioiodide requires a more efficient and specific system of gene delivery with better retention of radioiodide in tumor. Results thus far are, however, promising, and suggest that NIS gene transfer followed by radioiodide treatment will allow non-invasive in vivo imaging to assess the outcome of gene therapy and provide a therapeutic strategy for a variety of human diseases.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.12
• /
• pp.1781-1784
• /
• 2001

We have examined the influence of antisense oligo deoxynucleotide (ODN) of IGFBP-2 on tissue IGFBP-2 gene expression in chicks. Antisense IGFBP-2 ODN was directly injected into the liver or cerebroventricle. Control birds were injected with vehicle. The hepatic IGFBP-2 gene expression was decreased to approximately 30% of the control at 2 h after injection of antisense ODN. In the brain of chickens injected with antisense ODN, IGFBP-2 mRNA level did not change after 2 h of injection and decreased to approximately 60% of the control after 6 h of injection. These results showed that the expression of IGFBP-2 gene in the liver and brain was successfully suppressed by administrating antisense ODN and that hepatic IGFBP-2 gene expression was quickly suppressed by antisense ODN compared with the brain.