Design of a Real-Time Quantitative Polymerase Chain Reaction to Assess Human Complement Regulatory Protein Gene Expression in Polytransgenic Xenograft Pigs

• Published in: Transplantation proceedings Vol. 42; no. 8; pp. 3235 - 3238
• Main Authors: Martínez-Alarcón, L, Quereda, J.J, Herrero-Medrano, J.M, Majado, M.J, Mendonça, L, Pallarés, F.J, Ríos, A, Ramírez, P, Muñoz, A, Ramis, G
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier Inc 01.10.2010; Elsevier
• Subjects: Animals; Animals, Genetically Modified; Base Sequence; Biological and medical sciences; Complement System Proteins - metabolism; DNA Primers; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Male; Medical sciences; Polymerase Chain Reaction - methods; RNA, Messenger - genetics; Surgery; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Swine; Tissue, organ and graft immunology; Transcription, Genetic; Transplantation, Heterologous; Human; Complement regulatory protein; Complement; Transplantation; Gene expression; Heterograft; Heterotransplantation; Pig; Design; Medicine; Vertebrata; Goal; Mammalia; Treatment; Surgery; Animal; Graft; Artiodactyla; Ungulata; Quantitative analysis; Real time polymerase chain reaction
• ISSN: 0041-1345; 1873-2623
• DOI: 10.1016/j.transproceed.2010.05.062

Abstract Objective To design a real-time quantitative polymerase chain reaction (q-PCR) to assess gene expression for hCD55, hCD59, and hCD46 in polytransgenic (PT) pigs used as xenograft donors for orthotopic liver xenotransplantation using a pig-to-baboon model. Materials and methods Three pairs of primers were designed using PrimerBlast and mRNA of hCD55, hCD59, and hCD46 sequences. Blood samples from five PT pigs (two males and three females) were used to isolated peripheral blood mononuclear cells (PBMCs) by means of Ficoll gradients. After DNAase digestion of isolated mRNA, we synthesized cDNA. Using SYBR-Green chemistry of q-PCR, we constructed a standard curve. Two wild-type (WT) pigs were used as negative controls, and PBMCs from two healthy human volunteers as positive controls. The amplicon length was assessed by means of agarose gel electrophoresis and PCR products, sequenced. Results We observed amplification for hCD55, hCD59, and hCD46 in all samples from the five PT pigs except for hCD55 and hCD46 in one male PT pig. Neither the human samples nor the negative controls showed amplification. The expected amplicon length was confirmed; sequencing showed high homology with human mRNA for the three proteins and no match with any known pig sequence. Conclusions The q-PCR allowed detection of animals with the highest gene expression for hCD55, hCD59, and hCD46 for xenograft donors in transplantation experiments.