Rapid detection of human HLA transgenes in pigs by fluorescence in situ hybridization (FISH) for adjuvant study of human xenotransplantation.
OBJECTIVE
We have recently generated several lines of transgenic pigs for HLA-DP and -DQ to elucidate the role of HLA-II antigens in the modulation of cell-mediated rejection of xenotransplantation. Using fluorescence in situ hybridization (FISH) analysis, the aim of this study was to determine integration sites and to test zygosity of these transgenes in the piglets after cross mating.


METHODS
Blood lymphocytes of transgenic pigs for HLA-DP and -DQ were collected and cultured. Chromosome spreads were prepared by standard methodology. Gene constructs of HLA-DP A1+B1, -DQ A1 & B1 were labeled with fluorescein isothiocyanate or Texas Red by nick-translation. Hybridization was based on a standard FISH protocol.


RESULTS
FISH analysis revealed that the HLA-DP probe hybridized to porcine chromosome 6, while both HLA-DQ A1 and B1 probes hybridized to porcine chromosome 11 at the same site. There was no cross-hybridization of HLA transgenes to the swine leukocyte antigen complex. Mosaic integration of HLA-DQ transgenes in the genome of F0, but full penetrance in F1 after selective breeding was observed. Both HLA-DP and HLA-DQ lines were determined to be heterozygous at the integration site.


CONCLUSIONS
By FISH, we have detected specific integration sites of the HLA-DP and -DQ transgenes in pig genome and determined mosaic levels and zygosity types of these transgenes. We conclude that FISH is both sensitive and labor-efficient in confirming and differentiating transgenic pigs for multiple rejection-regulatory genes by visualizing individual integration sites in chromosomes or interphase nuclei.
Wang
Bao-Tyan
BT
Department of Medical Research, Changhua Christian Hospital, Changhua, Taiwan.
Tu
Ching-Fu
CF
Hsieh
Lie-Jiau
LJ
Tai
Hao-Chih
HC
Chiu
Ya-Lan
YL
Lee
Jang-Ming
JM
Kuo
Shou-Jen
SJ
Tsuji
Kimiyoshi
K
Lee
Chun-Jean
CJ
D000818:Animals; D030801:Animals, Genetically Modified; D000910:Antibodies, Heterophile / Q000097:blood; D006084:Graft Rejection / Q000276:immunology; D006680:HLA Antigens / Q000032:analysis / Q000235:genetics*; D006682:HLA-DP Antigens / Q000097:blood / Q000235:genetics; D006683:HLA-DQ Antigens / Q000097:blood / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology*
D000910:Antibodies, Heterophile; D006680:HLA Antigens; D006682:HLA-DP Antigens; D006683:HLA-DQ Antigens