• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.259-268
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• 2003

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinoma cells and is known to prevent symptomatic rotavirus infections. In this study, we tried to transfer the human lactadherin gene to the Chinese Hamster Ovary (CHO) cells using retrovirus vector system and tested inducible expression of the gene under the tetracycline-controllable promoter. At first, tetracycline-mediated inducibility was tested using E.coli LacZ marker gene. NIH3T3 cells co-infected with RevTet-On and RevTRE-LacZ retrovirus vectors showed that the cells responded to doxycycline (a derivative of tetracycline) in a dose-dependent manner, and prominent induction of the lacZ gene expression was observed from 1 $\mu\textrm{g}$/ml of doxycycline concentration. Based on the results of the pilot experiment, inductional expression of the human lactadherin gene was conducted using RevTet-On and RevTRE-Ltd retrovirus vectors. Analysis with the RT-PCR demonstrated successful inductional expression of the lactadherin gene in the Chinese Hamster Ovary (CHO) cells. Considering that constitutive overexpression of the exogenous genes in the target cells causes serious physiological imbalance, the results obtained in this study will be very useful especially in the studies of gene therapy and transgenic animal production.

• 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.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.15-20
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• 2010

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. Two classes of infectious human-tropic replication-competent PERVs (PERV-A and PERV-B) and one class of ecotropic PERV-C are known. The potential for recombination between ecotropic PERV-C and human-tropic PERVs adds another level of infectious risk. A recombinant PERV-A/C (PERV-A14/220) virus is 500-fold more infectious than PERV-A. Two determinants of this high infectivity was identified; one was isoleucine-to-valine substitution at position 140 in RBD (receptor binding domain), and the other lies within the PRR (proline rich region) of the envelope protein. To examine whether the effects of the cytoplasmic tail of the PERV-C Env on fusogenesity also influences infectivity, we constructed a pseudotype retroviral vectors containing MoMLV core protein and PERV envelopes. Pseudotyping experiments with the PERV envelope glycoproteins indicated that recombinant PERV-A/C virus is 10-fold more infectious than PERV-A by lacZ staining. This result supports the suggestion that viral transduction of PERV-A/C is enhanced by a membrane-proximal cytoplasmic amphiphilic ${\alpha}$-helix in PERV-C Env tail.