Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes

PLoS One. 2013 May 21;8(5):e63241. doi: 10.1371/journal.pone.0063241. Print 2013.

Abstract

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CD55 Antigens / genetics*
  • CD59 Antigens / genetics*
  • Cell Death
  • Cell Separation
  • Complement System Proteins / genetics*
  • Embryo, Mammalian / metabolism
  • Endothelial Cells / metabolism
  • Female
  • Fibroblasts / metabolism
  • Flow Cytometry
  • Fucosyltransferases / genetics*
  • Galactose / metabolism
  • Gene Expression
  • Gene Transfer Techniques*
  • Genetic Vectors / genetics
  • Humans
  • Immunohistochemistry
  • Male
  • Organ Specificity / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Serum
  • Swine / genetics*
  • Swine, Miniature / genetics*
  • Transgenes

Substances

  • CD55 Antigens
  • CD59 Antigens
  • RNA, Messenger
  • Complement System Proteins
  • Fucosyltransferases
  • Galactose

Grants and funding

This work was supported by a grant of Next-Generation BioGreen 21 Program (No. PJ00832303) Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.