Zink finger nucleases and siRNAs: use in transgenic pig

production for xenotransplantation

Heiner Niemann, Bjçrn Petersen, Hellen Ahrens,Wilfried Kues

Institute of Farm Animal Genetics (FLI), Mariensee, Neustadt, Germany

Emerging technologies, including the use ofspecific nucleases, transposons and specific siRNAswill bring significant improvements with regard toefficiency, precision and safety of gene transfer.Here, we summarize recent findings from ourlaboratory on the integration of Zinc-finger nucle-ase technology (ZFNs) and specific siRNAs into aresearch programme towards the development andcharacterization of multi-transgenic pigs suitablefor xenotransplantation.Zinc-finger nucleases (ZFNs) are powerful tools

for producing gene knockouts (KOs) with very highefficiency. ZFNs were used to induce a biallelicknockout of the porcine a1,3-galactosyltransferase(GGTA1) gene by employing primary porcinefibroblasts that were treated with ZFNs designedagainst the region coding for the catalytic core ofGGTA1 in SCNT. This resulted in biallelic knock-out of a high proportion of ZFN-treated cells. ZFN-mediated genetic modification did not interfere withthe cloning process. Off-target cleavage events orintegration of the ZFN-coding plasmid were notdetected. The absence of Gal epitopes was deter-mined by FACS and the GGTA1-KO phenotypewas confirmed by a complement lysis assay thatdemonstrated significant protection ofGGTA1-KOfibroblasts relative to wild-type cells. The ZFNswere functional irrespective of the sex of the donorcells, thus allowing the effective production offemale and male homozygous Gal kO pigs viasomatic cloning and the rapid establishment ofGal-/- pig lines. These pigs will serve as geneticbackground upon which additional transgenes canbe inserted and expressed.SiRNAs have emerged as useful tool to knock-

down specific genes in complex organisms for avariety of targets and serve as an important tool inthe functional analysis of specific genes. The acutevascular rejection (AVR) remains the main hurdlefor long term survival of a porcine xenograft aftertransplantation into primates. Immunological reac-

tions and molecular incompatibilities can lead toendothelial activation and microvascular thrombo-sis. Knock-down of tissue factor (TF) could be apromising mechanism to prevent AVR. TF is a keymolecule of the extrinsic coagulation pathway. Itfunctions as cell surface receptor for coagulationfactor VIIa and thereby initiates thrombin forma-tion. Since TF knock-out was lethal in the mousemodel, we tested different siRNAs in their efficiencyin a porcine cell line to knock-down TF. TwosiRNAs reduced the mRNA level of TF to <3%compared with wild type controls as determined byRT-PCR. Subsequently, porcine fetal fibroblasts(PFF) were co-transfected with constructs codingfor one of the promising siRNAs and a DsRedvector which confers neomycin resistance and redfluorescence. Transfected cells were selected withneomycin for 14 days and observed for the occur-rence of fluorescence. Integration of the siRNAvector was confirmed by PCR. Cell clones positivefor TF knock-down siRNA served as donor cells forsomatic cell nuclear transfer (SCNT). Recon-structed embryos were transferred to synchronisedrecipient sows. Eight transfers resulted in fourpregnancies. One foster animal was sacrificed onday 35 of gestation. Three fetuses were recoveredthat had integrated the TF knock-down siRNA andthe derived fetal cells were used for recloning andmore piglets were produced. The functionality of theknock-down is tested in a coagulation assay and inan established TNF alpha induced assay usingcultured endothelial cells of knock-down piglets incomparison with wild type counterparts. Exposureto inflammation mediators is known stimulateexpression of TF and adhesion molecules in theendothelium. Our genetically modified TF trans-genic pigs will be further characterized and pigs withthe desired TF knockdown will combined withalready existing genetic modifications to generatemulti-transgenic pigs. This model would be of greatimportance to prolong xenograft survival and moveporcine xenografts to the pre-clinical level.This project was funded by DFG (Ni 256/22-3 u.

4) and the EU project ‘‘Xenome’’ (LSHB-CT-2006-037377).

Berlin Symposium on Xenotransplantation 2012 – Abstracts

45

Xenotransplantation 2013: 20: 39–64Printed in Singapore. All rights reserveddoi: 10.1111/xen.12014

� 2013 John Wiley & Sons A/S.

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