232 GENERATION OF TRANSGENIC PIGS CARRYING A SMALL INTERFERING RNA VECTOR DIRECTED AGAINST TISSUE FACTOR EXPRESSION

Abstract

The acute vascular rejection (AVR) remains the main hurdle for long-term survival of a porcine xenograft after transplantation into primates. Immunological reactions and molecular incompatibilities can lead to endothelial activation and microvascular thrombosis. Knock-down of tissue factor (TF) could be a promising mechanism to prevent AVR. Tissue factor is a key molecule of the extrinsic coagulation pathway. It functions as cell surface receptor for coagulation factor VIIa and thereby initiates thrombin formation. Since TF knockout was lethal in the mouse model (Toomey et al. 1996 Blood 88, 1583–1587), we tested different small interfering (si) RNAs in their efficiency to knock down TF in a porcine cell line. Two siRNAs reduced the mRNA level of TF to <3% compared with wild type controls as determined by RT-PCR. Subsequently, porcine fetal fibroblasts (PFF) were co-transfected with constructs coding for one of the most promising siRNAs and a DsRed vector, which confers neomycin resistance and red fluorescence. Transfected cells were selected with neomycin for 14 days and checked for the occurrence of fluorescence. Integration of the siRNA vector was confirmed by PCR. Cell clones positive for TF knock-down siRNA served as donor cells for somatic cell nuclear transfer (SCNT). Successfully reconstructed embryos were transferred to synchronised recipient sows. Eight transfers resulted in 4 pregnancies. One foster animal was sacrificed on Day 35 of gestation. All of the 3 resulting fetuses had integrated the TF knock-down siRNA as shown by PCR and will be used for recloning. Additionally, we plan to determine vector integration by Southern blot analysis and siRNA and TF expression level by real time-PCR. The remaining 3 sows will be allowed to carry to full term for further investigations. The functionality of the knock-down will be tested in a tumo necrosis factor-α induced assay using cultured endothelial cells of knock-down piglets and of wild type counterparts because exposure to inflammation mediators stimulates expression of TF and adhesion molecules in the endothelium. Our genetically modified TF transgenic pigs can be combined with already existing genetic modifications to generate multi-transgenic pigs. This model would be of great importance to prolong xenograft survival and move porcine xenografts to the preclinical level.

This project was funded by the EU project ‘Xenome’ (LSHB-CT-2006-037377).