
%0 Journal Article
%A Kwon, M. S. 
%A Koo, B. C. 
%A Choi, B. R. 
%A Lee, H. 
%A Kim, T. 
%T 176 DOXYCYCLINE-INDUCIBLE EXPRESSION OF THE hEPO GENE IN A RETROVIRUS VECTOR
%J Reproduction, Fertility and Development
%D 2007/12/12
%V 20
%N 1
%P 167-168
%K 
%R https://doi.org/10.1071/RDV20N1AB176
%U https://www.publish.csiro.au/paper/RDV20N1AB176 
%X The use of &apos;bioreactors&apos; to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to medical advancement. However, it is well known that constitutive overexpression of some cytokine genes may cause serious physiological disturbances in vivo and in vitro. The main objective of this study was to test the feasibility of controllable gene expression of hEPO, a glycoprotein hormone that stimulates red blood cell production. We constructed and tested retrovirus vectors designed to express the hEPO (human erythropoietin) gene under the control of tetracycline-inducible promoters. Advantages of this system over other counterparts, whose gene expressions are controlled by steroid hormones or heavy metals, are little or no cytotoxicity of the inducer and efficient turn-on/off transition. However, the most critical problem of the tetracycline-controllable gene expression system is that overall gene expression level is low. As a solution for this problem, we introduced a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence into the retrovirus vector at a downstream region of either the hEPO gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator) of Tet-On system. In cooperation with tetracycline, the rtTA activates the tetracycline response promoter which controls the expression of the hEPO gene. Transformed cells derived from pig and chicken were cultured in medium supplemented with or without 1 &#181;g mL&#8211;1 of doxycycline (a tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the hEPO gene expression level using RT-PCR, Western blotting, and ELISA. Western blotting analysis showed that, among several vectors tested, the vector carrying the WPRE sequence at the 32 side of the hEPO gene was the best in both chicken and porcine cells. Quantitative analysis using ELISA resulted in a more than 10-fold increase in overall hEPO gene expression without sacrificing the efficiency of turn-on/off transition. These results should be very helpful in establishing transgenic livestock as bioreactors, making it possible to produce commercially valuable and biologically active proteins in large quantities. 
