224 TETRACYCLINE-INDUCIBLE GENE EXPRESSION WITH RETROVIRUS VECTOR

Abstract

One of the critical problems to be solved in transgenic animal production is non-controllable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP (green fluorescent protein) gene under the control of tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence was also introduced into the retrovirus vector downstream of either the GFP gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator). Transformed cells derived from porcine fetus were cultured in the medium supplemented with or without doxycycline (tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at the 3′ side of the GFP gene, while tighter expression control (up to 20-fold) was obtained from the vector in which the WPRE sequence was placed at the 3′ side of the rtTA sequence. Encouraged with these data, we substituted the hPTH (human parathyroid hormone) gene for the GFP gene in the retrovirus vector. The porcine fetal fibroblast cells transformed by the modified retrovirus vector secreted hPTH into the medium under the tight control of doxycycline as observed in GFP expression. The resulting porcine cells secreting hPTH will be used in nuclear transfer experiment. This study was financially supported by the National Livestock Research Institute RDA (Suwon 441-350, Korea), ARPC (Agriculture R & D Promotion center, 2002–2005), and by grant No. R11-2002-100-01000-0 from the ERC program of the Korea Science & Engineering Foundation.