Mo-Sun Kwon

Production of Pigs Expressing a Transgene under the Control of a Tetracycline-Inducible System

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.

38 PRODUCTION OF TRANSGENIC DOGS THAT OVEREXPRESS PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-ALPHA IN A MUSCLE-SPECIFIC MANNER

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Among 3 major types of PPAR (PPARα, PPARβ, and PPARγ), PPARα is expressed in tissues having a high rate of β-oxidation, such as brown adipose tissue, liver, kidney, heart, and skeletal muscle. The purpose of this study was to produce transgenic dogs that overexpress PPARα in a muscle-specific manner. Male beagle fetal fibroblasts were infected with viruses containing PPARα under a human myoglobin promoter and green fluorescent protein (GFP) under an EF1 promoter. Stable infectants were isolated with puromycin, and stored in liquid nitrogen until somatic cell nuclear transfer. Natural oestrus and ovulation of 22 dogs (13 oocyte donors and 9 recipients) was monitored by serum progesterone concentration, and in vivo-matured oocytes were recovered by surgical oviduct flushing. After cumulus cells were removed by repeated pipetting, nuclear materials of the denuded oocytes were removed. A GFP-expressing cell was selected under ultraviolet light, and injected into the enucleated oocyte. Then, the oocyte-cell couplets were fused electrically and activated chemically. Immediately after activation, the cloned embryos were transferred into the oviducts of recipient dogs. Pregnancy diagnosis was performed at least 27 days after embryo transfer by ultrasonography, and serum progesterone concentration, rectal temperature, and fetal heartbeat were monitored for safe delivery. As results, 152in vivo oocytes were recovered from oocyte donor dogs, and 116 cloned embryos were produced with the infected fetal fibroblast cells. Among them, 113 cloned embryos were transferred into 9 recipient dogs, and 3 were diagnosed as pregnant. Three healthy transgenic cloned puppies were produced, and they could serve as a canine model having enhanced physical performance and obesity resistance.

Production of hTPO Transgenic Chickens using Tetracycline-Inducible Expression System

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions (rtTA2 S M2) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins. (Key words : tetracycline-inducible expression system, transgenic chickens, human thrombopoietin (hTPO), bioreactor)