Zink finger nucleases and siRNAs: use in transgenic pig production for xenotransplantation

Emerging technologies, including the use of specific nucleases, transposons and specific siRNAs will bring significant improvements with regard to efficiency, precision and safety of gene transfer. Here, we summarize recent findings from our laboratory on the integration of Zinc-finger nuclease technology (ZFNs) and specific siRNAs into a research programme towards the development and characterization of multi-transgenic pigs suitable for xenotransplantation. Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with very high efficiency. ZFNs were used to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene by employing primary porcine fibroblasts that were treated with ZFNs designed against the region coding for the catalytic core of GGTA1 in SCNT. This resulted in biallelic knockout of a high proportion of ZFN-treated cells. ZFN-mediated genetic modification did not interfere with the cloning process. Off-target cleavage events or integration of the ZFN-coding plasmid were not detected. The absence of Gal epitopes was determined by FACS and the GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated significant protection of GGTA1-KO fibroblasts relative to wild-type cells. The ZFNs were functional irrespective of the sex of the donor cells, thus allowing the effective production of female and male homozygous Gal kO pigs via somatic cloning and the rapid establishment of Gal-/- pig lines. These pigs will serve as genetic background upon which additional transgenes can be inserted and expressed. SiRNAs have emerged as useful tool to knockdown specific genes in complex organisms for a variety of targets and serve as an important tool in the functional analysis of specific genes. 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. TF 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 knock-out was lethal in the mouse model, we tested different siRNAs in their efficiency in a porcine cell line to knock-down TF. 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 promising siRNAs and a DsRed vector which confers neomycin resistance and red fluorescence. Transfected cells were selected with neomycin for 14 days and observed 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). Reconstructed embryos were transferred to synchronised recipient sows. Eight transfers resulted in four pregnancies. One foster animal was sacrificed on day 35 of gestation. Three fetuses were recovered that had integrated the TF knock-down siRNA and the derived fetal cells were used for recloning and more piglets were produced. The functionality of the knock-down is tested in a coagulation assay and in an established TNF alpha induced assay using cultured endothelial cells of knock-down piglets in comparison with wild type counterparts. Exposure to inflammation mediators is known stimulate expression of TF and adhesion molecules in the endothelium. Our genetically modified TF transgenic pigs will be further characterized and pigs with the desired TF knockdown will 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 pre-clinical level.