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Successful production and breeding of cloned pigs expressing human thrombomodulin in endothelial cells

Masaki Iwamoto^1,2, Satoko Yazaki^1,2, Akira Onishi², Yuko Miwa³, Michiko Hashimoto¹, Takatsugu Oishi¹, Shunichi Suzuki², Dai-ichiro Fuchimoto², Shoichiro Sembon², Takaharu Nagasaka³, DaGe Liu³, Kenta Iwasaki³, Masataka Haneda³, Koji Yamamoto³, Shoichi Maruyama³, Takaaki Kobayashi³

¹Prime Tech Ltd, Tsuchiura; ²National Institute of Agrobiological Sciences, Tsukuba; ³Nagoya University School of Medicine, Nagoya; Japan

Introduction: Coagulation control is one of the remaining critical issues for long-term xenograft survival. Human thrombomodulin (hTM) is expected to exhibit the beneficial effect on coagulation control, because hTM could (i) solve the problem of molecular incompatibility in protein C activation, (ii) exert a role as a physiological regulator, only when thrombin is formed, (iii) suppress direct prothrombinase activity and (iv) have anti-inflammatory properties. We attempted to produce cloned pigs expressing hTM.

Methods: pCAGGS/hTM expression vector was transfected into pig (Landrace/Yorkshire) fibroblasts. After puromycin selection for 7 days, surviving cells were stained with mouse anti-hTM antibody and FITC-labeled goat anti-mouse IgG antibody. hTM-expressing cells at high levels were collected by gating on fluorescence intensity, and were applied to nuclear transfer. After electoractivation and subsequent culture, the cleaved embryos were transferred to 7 surrogate mother pigs.

Results: Two cloned piglets expressing hTM were safely born. The progeny were successfully produced and showed no problems in growth rate or in sexual maturation. Aortic endothelial cells from cloned pigs (hTM-PAEC) were found to express hTM at comparable levels to HUVEC. hTM-PAEC showed a significant level of APC production, improvement of clotting time, suppression of thrombin formation and anti-inflammatory function. Immunohistochemical staining of kidney, liver and heart from hTM pigs demonstrated hTM expression in endothelial cells.

Conclusions: Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were successfully produced. Cross-breeding with other genetically modified pigs is now in progress.