56 PRODUCTION OF TRANSGENIC RECLONED MINIATURE PIGS EXPRESSING HUMAN DECAY ACCELERATING FACTOR

Abstract

This study was performed to produce transgenic recloned miniature pigs expressing human decay-accelerating factor (hDAF) to overcome the hyperacute rejection of pig-to-human xenotransplantation. The expression vector, named hDAF-Neo, was constructed by subcloning the amplified 2.2 kb hDAF cDNA fragment into the NheI and NotI site of the pEGFP-N1 (Clontech, Palo Alto, CA, USA) vector containing the chicken beta-actin promoter and rabbit globin poly A without a EGFP fragment. Day 30 fetal fibroblast cells (GN0) were used for producing the newborn cloned pigs (GN1). GN1 fibroblasts cultured from the newborn cloned pig derived from GN0 were used for transfection of the hDAF-Neo plasmid using FuGENE-6^® (Roche Diagnostics, Indianapolis, IN, USA). Transfected fibroblast cell colonies were selected with neomycin. All data were analyzed by one-way ANOVA and the protected least significant difference (LSD) test using general linear models in a statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Significant difference among the treatment groups was determined when the P value was less than 0.05. There was no significantly different development rate between GN0 and GN1 (12.8 vs. 13.0%) and between GN1-hDAF and GN2 (9.3 vs. 8.2%). Transfected recloned embryos (GN1-hDAF) had no significantly different blastocyst formation compared to normal (GN1). After embryo transfer, we obtained two transgenic recloned pigs. hDAF gene integration in transgenic recloned piglets was confirmed by polymerase chain reaction. Cultured fibroblasts of transgenic recloned pigs showed 1.0-2.3 times higher levels of hDAF protein than did human umbilical vein endothelial cells. The levels of C3 deposition on cultured fibroblast cells after incubation with 10% human serum were decreased in transgenic recloned pigs compared to normal miniature pigs. In conclusion, the recloning procedure can be used to produce multiple genetically modified cloned pigs without any severe epigenetic abnormality.

This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).