title: siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.
abstract: Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, Neustadt, Germany.
authors: H E
authors: HE
authors: Ahrens
authors: B
authors: B
authors: Petersen
authors: D
authors: D
authors: Herrmann
authors: A
authors: A
authors: Lucas-Hahn
authors: P
authors: P
authors: Hassel
authors: M
authors: M
authors: Ziegler
authors: W A
authors: WA
authors: Kues
authors: U
authors: U
authors: Baulain
authors: W
authors: W
authors: Baars
authors: R
authors: R
authors: Schwinzer
authors: J
authors: J
authors: Denner
authors: D
authors: D
authors: Rataj
authors: S
authors: S
authors: Werwitzke
authors: A
authors: A
authors: Tiede
authors: A K
authors: AK
authors: Bongoni
authors: P S
authors: PS
authors: Garimella
authors: A
authors: A
authors: Despont
authors: R
authors: R
authors: Rieben
authors: H
authors: H
authors: Niemann
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001777:Blood Coagulation; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism; D005821:Genetic Techniques; D006084:Graft Rejection; D006801:Humans; D008297:Male; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D034421:Sus scrofa; D013737:Testis / Q000166:cytology; D013925:Thromboplastin / Q000378:metabolism*; D013927:Thrombosis / Q000473:pathology*; D014183:Transplantation, Heterologous
chemical_list: D034741:RNA, Small Interfering; D013925:Thromboplastin
keywords: animal models: porcine; basic (laboratory) research / science; cloning; coagulation and hemostasis; genetics; molecular biology; rejection: vascular; xenotransplantation