192 INDUCIBLE TRANSGENE EXPRESSION IN PIGS

Abstract

Genetically modified pigs are important models for biomedical research. For certain applications, tightly controlled transgene expression is mandatory. For instance, expression of soluble receptor activator of the NFκB ligand (RANKL) is an established principle for inducing clinically relevant osteoporosis (Mizuno et al. 2002 J. Bone Miner. Metab. 20, 337–344). However, constitutive overexpression of RANKL is lethal; thus, an inducible system is required. A second example is the expression of porcine CTLA-4Ig to prevent co-stimulation of T-cells in pig-to-primate xenotransplantation. Because constitutive expression of porcine CTLA-4Ig causes immunosuppression in pigs (Phelps et al. 2009 Xenotransplantation 16, 477–485), an inducible expression system is desirable. The Tet-On system is used most widely. It is based on a tetracycline transactivator (TA) and a transactivator response element (TARE), which drives expression of the coding sequence for the transgene product of choice. Association of doxycyclin with TA allows binding to TARE, resulting in activation of transgene expression. Although widely used in mice, this system has not yet been established in pigs. Because consistently high expression of TA is required for a functional system, we first cloned an expression vector for TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). A floxed neomycin resistance cassette was linked to this vector to facilitate selection of stable clones after transfection of primary porcine kidney cells. The latter were used for nuclear transfer, and cloned embryos were transferred to synchronized recipient gilts, resulting in the birth of 5 CAG-TA transgenic piglets. A spectrum of organs and tissues was investigated by RT-PCR and Western blot for TA mRNA and protein expression. Primary kidney cells from all 5 TA transgenic piglets were transfected with TARE-RANKL and TARE-CTLA-4Ig constructs linked with a blasticidin resistance cassette to facilitate selection of double transgenic/transfected cell clones. These cell clones were individually stimulated with doxycyclin to identify cell clones with low background and high inducibility of expression of RANKL or CTLA-4Ig. For both combinations, several clones meeting these criteria were identified. Such cell clones were used for nuclear transfer to produce double-transgenic pigs. Application of doxycyclin (25 to 50 mg kg^–1 of body weight/day) to a TA/TARE-RANKL transgenic pig resulted in a marked increase in plasma RANKL concentrations. In addition, 4 TA/TARE-CTLA-4Ig transgenic piglets have been born, which will undergo doxycyclin stimulation experiments in the near future. In summary, CAG-TA transgenic pigs serve as a reliable basis for inducible transgene expression, as shown in the present study for RANKL and CTLA-4Ig. Studies to show the biological functionality of the transgene are currently underway.