Rapid Detection of Human HLA Transgenes in Pigs by Fluorescence in Situ Hybridization (FISH) for Adjuvant Study of Human Xenotransplantation

10.30047/JGMB.200603.0005

Bao-Tyan Wanq；Ching-Fu Tu；Lie-Jiau Hsieh；Hao-Chih Tai；Ya-Lan Chiu；Jang-Ming Lee；Shou-Jen Kuo；Kimiyoshi Tsuji；Chun-Jean Lee

fluorescence in situ hybridization ； HLA transgenes ； xenotiansplantation

Journal of Genetics and Molecular Biology

17卷1期（2006 / 03 / 01）

20 - 26

英文

Objectives: 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 H LA-DP A1+B1, -DQ Al & B1 were labeled with fluorescein isothiocvanate 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 Al and B1 probes hybridized to porcine chromosome Ⅱ 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. Conclusion: 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 rejecti on-regulatory genes by visualizing individual integration sites in chromosomes or interphase nuclei.