• Clinical and Experimental Pediatrics
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• v.50 no.10
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• pp.1011-1017
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• 2007

Purpose : We tried to assess the optimal conditions to improve low transduction efficiency and their effect on target cells. Methods : Cultured NIH 3T3 cells were incubated with retroviral vectors bearing an enhanced green fluorescent protein (eGFP) gene. We varied the ratio of viral vectors to target cells (1:1-1:8) and the number of transfections (${\times}1$, ${\times}2$), and compared transduction efficiencies. Also, the effects of polybrene on transduction efficiency and viability of target cells were assessed. Transduction of the eGFP gene was evaluated by observing NIH 3T3 cells under a fluorescence microscope and efficiencies were measured by the percentage of eGFP positive cells using FACscan. Results : As the ratio of retroviral vectors to target cells increased, transduction efficiency was greatly improved, from 7% (1:1) to 38% (1:4). However, transduction efficiency did not increase any more when the ratio increased from 1:4 to 1:8. Cells transfected twice showed higher transduction efficiencies than cells transfected once, at a ratio of 1:8. The eGFP gene transduced to NIH 3T3 cells sustained its expression during repeated passages. However, after the third passage (day 9), the percentage of eGFP positive cells began to decline. The degree of this decline in eGFP expression was lower in cells transfected twice than in cells transfected once (P<0.05). The addition of polybrene did not have any toxic effect on NIH 3T3 cells and greatly increased transduction efficiency (P=0.007). In addition to vector component, transduction efficiency was very sensitive to culture confluence. Cells cultured and transfected in 24-well plate showed higher transduction efficiency, although cells cultured in 6- well plate proliferated more (P=0.024). Conclusion : Our data could be used as a basis for retrovirus-based gene therapy. Further study will follow using human cells as target cells.

• KSBB Journal
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• v.14 no.5
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• pp.593-599
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• 1999

In this study, optimal conditions to infect CD34 positive cells containing hematopoietic stem cells obtained from cord blood and bone marrow were found using two different retroviral vectors expressing human growth hormone (hGH) and $\beta$-galactosidase. CD34 positive cells were successfully infected with recombinant retroviruses only when the CD34 positive cells were co-cultured with packaging cells secreting recombinant retroviruses. To find the highest infection efficiency for the gene transfer, CD34 positive cells from cord blood were co-cultured with packaging cells secreting recombinant retroviruses encoding E. coli lacZ gene. The highest infection efficiency was obtained when CD34 positive cells were cultured for 3 days, and then co-culturing was done for another 2 days. When CD34 positive cells from bone marrow were co-cultured with packaging cells secreting recombinant retroviruses encoding hGH gene, the maximum amount of hGH was also secreted at the same conditions found above, i.e. 3 days of culture and 2 days of co-culture. These results show that there are optimal conditions for the gene transfer into hematopoietic stem cells regardless of sources of target cells or retroviral vectors used to infect.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.3
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• pp.226-233
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• 2007

Purpose: Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. Materials and Methods: A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. Results: The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, $T_{1/2}$ of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. Conclusion: A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene.

• Tuberculosis and Respiratory Diseases
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• v.44 no.1
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• pp.162-174
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• 1997

Background : Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hyporthesized that treatment of tumor cells with retinoic acid could augment the bystander effect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. Methods : We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gene to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of tumor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. Results : Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin 43 and was asspciated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no delectable connexin 43. Conclusion : These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.

• Tuberculosis and Respiratory Diseases
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• v.41 no.2
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• pp.87-96
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• 1994

Background : Since tumor necrosis factor was discovered in 1975, TNF has been well known about its cytotoxic effect on tumor cells in vivo and in vitro. According to the recent improvement of molecular biological techinques, it is possible that exogenous TNF gene is transferred to tumor cells and is expressed in theirs. By virtue of TNF gene transfer, we have expected that TNF expressed in TNF-gene-transferred tumor cells would kill tumor cells in vivo without systemic side effect. The expected mechanisms in which antitumor effects of TNF expressed in TNF-gene-transferred tumor cells are working would be as followings. In the first mechanism, TNF expressed in TNF-gene-transferred tumor cells would kill tumor cells around(like homicide). In the second mechanism, TNF expressed in TNF-gene-transferred tumor cells would kill themselves(like suicide). In the third mechanism, TNF expressed in TNF-gene-transferred tumor cells would recruit immune effector cells and kill tumor cells indirectly. In the last mechanism, TNF expressed in TNF-gene-transferred tumor cells would augment cytokine such as interferon-$\gamma$ to kill tumor cells. Among these four mechanisms of antitumor effect, only the second mechanism has not been established yet. Therefore, to elucidate the second mechanism, We performed this study. Method : We transferred TNF-$\alpha$ gene to NCI-H2058, a human mesothelioma cell line and WEHI164, a murine fibrosarcoma cell line by using retroviral vector(pLT12SNTNF). And, We determined by using MTT assay whether TNF expressed in TNF-gene-transferred tumor cell lines would kill themselves like suicide or not. Then, if TNF-gene-transferred tumor cell lines would not suicide themselves, I would know more about the TNF sensitivity of TNF-gene-transferred tumor cell lines to exogenous TNF also by MTT assay. Result : NCI-H2058 and WEHI164 which were sensitive to TNF, became far less sensitive to endogenous and exogenous TNF after being transferred TNF-$\alpha$ gene to. Conclusion : TNF-gene-transfer to NCI-H2058 and WEHI164 gave them resistance to TNF.

• Tuberculosis and Respiratory Diseases
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• v.42 no.3
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• pp.302-313
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• 1995

Background: Tumor necrosis factor(TNF) showed antitumor cytolytic effects on sensitive tumor cells in numerous in vivo and in vitro studies. But it could not be administered systemically to human because of severe systemic adverse effects at effective concentrations against tumor cells. Many studies showed that a high concentrations of TNF in the local milieu may evoke in vivo TNF-responsive mechanisms sufficient to suppress tumor growth. Recently developed technique of TNF gene transfer to tumor cells using retrovirus vector could be a good candidate for local TNF administration. TNF is also known to synergistically enhance in vitro cytotoxicity of chemotherapeutic drugs targeted to DNA topoisomerase II against TNF-sensitive tumor cell lines. In this study the in vitro chemosensitivity against DNA topoisomerase II targeted chemotherapeutic drugs was evaluated using some respiratory cancer cell lines to which TNF gene had been transferred. Method: NCI-H2058, a human mesothelioma cell line, A549, a human lung adenocarcinoma cell line and WEHI 164 cell line, a murine fibrosarcoma cell line were treated with etoposide and doxorubicin, which are typical topoisomerase II - targeted chemotherapeutic agents, at different concentration. The resultant cytotoxicity was measured by MIT assay. Then the cytotoxicity of the same chemotherapeutic agents was measured after TNF-$\alpha$ gene-transfer and the two results were compared. Results: The cytotoxicity was not increased significantly in WEHI164 cell line and A549 cell line but statistically significant increase was observed in H2058 cell line when TNF-$\alpha$ gene was transferred(p<0.05). Conclusion: These findings show that TNF-$\alpha$ gene transfer to respiratory cancer cell lines results in variable effects on chemosensitivity against topoisomerase II inhibitor among different cell lines in vitro and can be additively cytotoxic in certain selective tumor cell lines.