title: Production of cloned pigs expressing human thrombomodulin in endothelial cells.
abstract: For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.
authors.affiliation: Prime Tech Ltd, Tsuchiura, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Yazaki
authors.forename: Masaki
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Akira
authors.initials: A
authors.lastname: Onishi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Miwa
authors.forename: Michiko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Oishi
authors.forename: Shunichi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Dai-ichiro
authors.initials: D
authors.lastname: Fuchimoto
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Sembon
authors.forename: Tadashi
authors.initials: T
authors.lastname: Furusawa
authors.forename: DaGe
authors.initials: D
authors.lastname: Liu
authors.forename: Takaharu
authors.initials: T
authors.lastname: Nagasaka
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kuzuya
authors.forename: Haruko
authors.initials: H
authors.lastname: Ogawa
authors.forename: Koji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Kenta
authors.initials: K
authors.lastname: Iwasaki
authors.forename: Masataka
authors.initials: M
authors.lastname: Haneda
authors.forename: Shoichi
authors.initials: S
authors.lastname: Maruyama
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001773:Blood Cells / Q000378:metabolism; D001777:Blood Coagulation; D019976:Cloning, Organism / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005818:Genetic Engineering; D006085:Graft Survival; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006824:Hybridization, Genetic; D007150:Immunohistochemistry; D008297:Male; D010314:Partial Thromboplastin Time; D011247:Pregnancy; D011486:Protein C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000097:blood / Q000235:genetics; D034421:Sus scrofa / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D018180:Thrombomodulin / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics*; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
chemical_list: D017931:DNA Primers; D011486:Protein C; D012333:RNA, Messenger; D011994:Recombinant Proteins; C546931:THBD protein, human; D018180:Thrombomodulin