• Journal of Aquaculture
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• v.12 no.1
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• pp.71-77
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• 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 Aquaculture
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• v.10 no.4
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• pp.409-415
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• 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.

• Journal of Aquaculture
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• v.12 no.4
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• pp.275-281
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• 1999

The utility of RSV-LTR and carp beta-actin promoters was evaluated in a marine flatfish species, Limanda Yokohamae by examining successful expression of transgenic DNA in muscles (transfected by direct injection) and in early embryos (transformed by lipofected sperm). The expressed pattern of injected DNA in skeletal muscles was dependent on the DNA amount injected. The activity reached to maximal level at 48 hours post injection, and persisted up ot 1 month transiently. Gene transfer into early embryo of this species was successfully achieved using lipofected sperm with the efficiency ranging 36.8% to 48.1%. The expression of transgene during embryonic development was shown as stage-specific and transient.

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

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

• KSBB Journal
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• v.19 no.5
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• pp.341-347
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• 2004

Several gene delivery therapies are being developed for treatment of serum protein deficiency. EPO is one of the most promising therapeutic agent for this treatment which is currently being investigated in depth. This study has the ultimate purpose of improving the gene delivery system for an increase of red blood cell production. A plasmid DNA was constructed smaller than other plasmids for an increase in penetration into animal cells, and two genes were cloned into each vector as a co-delivery system to express erythropoietin, and interluekin-3 or thrombopoietin, which can act on erythroid cell, thus activating hematopoiesis synergically. This co-delivery system has an advantage of decreasing the labour required for industrial production of DNA vaccine. A new plasmid vector, pVAC, in size 2.9 kb, was constructed with the essential parts from PUC 19 and pSectagB, which is much smaller than other plasmid vector and is the size of 2.9 kb. Co-delivery system was constituted by cloning human erythropoietin with each of human interluekin-3 gene or human thrombopoietin gene into both pVAC and pSectagB. As a result, the transfection efficiency of pVAC was higer than that of pSectagB in vitro, and hematocrit level of the mice injected with pVAC is higher than that of other mice. And co-delivery system, made of several plasmid DNAs, was expressed in vitro.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.145-152
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• 1998

Many trials have been made to produce transgenic animals using sperm cells as a vector transferring foreign DNA into eggs, but reliable results are yet to be obtained (Brinster et al., 1989; Lavitrano et al., 1989; Bachiller et al., 1991; Sato et al., 1994). Recently, one of author(SO) demonstrated that mouse blastocysts derived from eggs fertilized by spermatozoa of male mice single injected with liposome-DNA complexes within the testis expressed thegene (Ogawa et al., 1995.) Here we report that a single injection of liposome-encapsulated DNAs into the testis of either male rats or mice resulted in successfully gene transfer to the postpartum progeny. The expression of mRNA derived from transgenes was also demonstrated in transgenic animals thus obtained. Further, the transmission of the exogenous gene to the descedants was confirmed in one line of transgenic rat up to F4 generation, indicating that the gene was stably incorporated into the germ line. Thus, direct single injection of foreign DNA into the testis provides a novel and convenient means to generate transgenic animals.

• YAKHAK HOEJI
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• v.45 no.1
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• pp.108-115
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• 2001

We have recently reported the development of a high efficiency expression vector, pCK, which can drive a high level of gene expression in mouse skeletal muscle. In this study, we tested the therapeutic potential of pCK expressing human VEGF165, pCK-VEGF in the rabbit ischemic hind limb model. To determine the optimal dose of plasmid DNA, various concentrations of pCK-CAT were injected into the muscle of a rabbit hind limb and the levels of CAT activity were determined. It was found that the expression level of the exogenously added gene became stable between 250 and 1,000 $\mu$g. Based on this result, we tested whether intramuscular transfer of 500$\mu$g of pCK-VEGF could actually modulate collateral vessel development in a rabbit ischemic hind limb model. It was found that relative to the control group injected with the pCK lacking the VEGF sequence, single intramuscular doses (500$\mu$g) of pCK-VEGF produced statistically significant augmentation of collateral vessels as determined by the angiographic vessel count, maximal blood flow by Doppler flowmeter and the number of capillaries by histology. These results suggest that a single 500$\mu$g-delivery of pCK-VEGF is potent enough to induce sufficient angiogenic activity and achieve therapeutic benefit on this rabbit model.

• Asian-Australasian Journal of Animal Sciences
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• v.7 no.3
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• pp.427-434
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• 1994

The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.416.2-416.2
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• 2002

Transfersomes are highly detormable hydrophilic lipid vesicles that are able to penetrate the skin barrier so that they can be used to carry low- and high-molecular weight molecules into the body. Until recently. it has been reported that molecules such as insulin. interleukin-2 and several other large molecules have been transported into the body using Transfersomes as a delivery system. Here however, for the very first time, genes (GFP) have been transported into the mice non-invasively using the Transtersomes as a delivery vehicle. (omitted)