Design of a real-time quantitative polymerase chain reaction to assess human complement regulatory protein gene expression in polytransgenic xenograft pigs.
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.


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.
Hospital Universitario Virgen de la Arrixaca, Murcia, Spain.
L
L
Martínez-Alarcón
J J
JJ
Quereda
J M
JM
Herrero-Medrano
M J
MJ
Majado
L
L
Mendonça
F J
FJ
Pallarés
A
A
Ríos
P
P
Ramírez
A
A
Muñoz
G
G
Ramis
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003165:Complement System Proteins / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine; D014158:Transcription, Genetic; D014183:Transplantation, Heterologous
D017931:DNA Primers; D012333:RNA, Messenger; D003165:Complement System Proteins