Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.
Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p < 0.05). Caspase-3/-7 activity of the shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p < 0.05). These results show that shTNFRI-Fc and HA-hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury. Utilization of the F2A self-cleaving peptide is a promising tool for generating multiple TG pigs for xenotransplantation.
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-742, Korea.
Sol Ji
SJ
Park
Bumrae
B
Cho
Ok Jae
OJ
Koo
Hwajung
H
Kim
Jung Taek
JT
Kang
Sunghoon
S
Hurh
Su Jin
SJ
Kim
Hye Jung
HJ
Yeom
Joonho
J
Moon
Eun Mi
EM
Lee
Ji Yei
JY
Choi
Ju Ho
JH
Hong
Goo
G
Jang
Joing-Ik
JI
Hwang
Jaeseok
J
Yang
Byeong Chun
BC
Lee
Curie
C
Ahn
D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D010455:Peptides / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000096:biosynthesis* / Q000235:genetics; D034421:Sus scrofa; D013552:Swine / Q000235:genetics
C540800:F2A4-K-NS peptide; D007141:Immunoglobulin Fc Fragments; D010455:Peptides; D047888:Receptors, Tumor Necrosis Factor, Type I; D051547:Heme Oxygenase-1