First inducible transgene expression in porcine large animal models.

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken ß-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-κB ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.

Vascular incorporation of endothelial colony-forming cells is essential for functional recovery of murine ischemic tissue following cell therapy.

OBJECTIVE: Cord blood-derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell-based therapy, including mesenchymal stem cells (MSCs). METHODS AND RESULTS: ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor(+) cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. CONCLUSIONS: Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell-based therapy represents a promising approach for vascular regeneration of ischemic tissue.

Molecular cloning and functional characterization of the porcine extracellular domain of Receptor Activator of NF-κB Ligand (sRANKL).

Receptor Activator of NF-κB Ligand (RANKL) plays a pivotal role as a regulator of osteoclast activity and is involved in osteoporosis. Here, we report the cloning and functional characterization of the complete extracellular domain of the porcine RANKL gene (sRANKL). The porcine sRANKL cDNA has an ORF of 744 nucleotides and shares 87%, 80% and 80% identity with human, rat and mouse RANKL coding sequences, respectively. The protein consists of 247 amino acids with 90%, 81% and 80% sequences similarities compared to human, mouse and rat RANKL, respectively. Over-expression of porcine sRANKL led to osteoclast formation. The osteoclasts showed a characteristic morphology, expressed the carbonic anhydrase type 2, were TRACP positive and exhibited a bone-resorbing activity. In conclusion, we first describe the molecular cloning and functional characterization of porcine sRANKL, which will help to understand the function of a RANKL gene in large animal models.