20 PRODUCTION OF NUCLEAR TRANSFER EMBRYOS FROM PORCINE FETAL FIBROBLAST CELLS CARRYING A TETRACYCLINE-INDUCIBLE TRANSGENE

Abstract

Constitutive expression of A transgene often results in serious physiological disturbances in transgenic animals. For instance, systemic overexpression of human growth hormone in transgenic pigs has resulted in detrimental side effects in general health and reproductive performance. One of the solutions to such problem would be inducible expression of a transgene that may restrict production of foreign proteins from transgenic animals only when needed. In this study, a retrovirus vector was designed to express the green fluorescent protein (GFP) gene under the control of the tetracycline-inducible promoter. Transformation of porcine fetal fibroblast cells was achieved by infection of the cells with the vector and subsequent antibiotic selection. To induce transgene expression, transformed porcine fetal fibroblast cells were cultured in medium supplemented with doxycycline for 48 h. Induction of the GFP gene was verified by the emission of fluorescence from transformed cells. Nuclei of transformed cells with or without doxycycline treatment were transferred into enucleated oocytes, and the induction efficiency was analyzed by monitoring fluorescent emission during development of reconstituted embryos to the blastocyst stage. In addition, differences in the rates of blastocyst development between experimental groups were analyzed by Student's t-test. Blastocyst formation of nuclear transfer embryos using transformed cells with tetracycline-inducible retrovirus vector (12.0%, 128/1072) was not significantly different (P > 0.05) from that with non-inducible control vectors (13.7%, 41/300), suggesting that an introduction of tetracycline-inducible retrovirus vector was not particularly harmful to the development of nuclear transfer embryos. Also, the blastocyst development rate of nuclear transfer embryos after induction of transgene by doxycycline (12.1%, 99/815) was not significantly different (P > 0.05) from that of the non-induced counterparts (11.3%, 29/257), suggesting that the induction of transgene did not affect the development of transgenic clone embryos. In a majority of embryos, high expression of the GFP gene was observed in cloned embryos with transgene induction, whereas poor or no GFP expression was detected in non-induced controls. The results from this study suggest that tetracycline-inducible expression of transgenes in nuclear transfer embryos may be used for production of foreign proteins in transgenic animals in a more controlled manner than with conventional procedures. Further experiments on transfer of cloned embryos carrying such an inducible transgene to recipients may enable production of transgenic pigs with fewer side effects from unregulated expression of the transgene.