<abstract xmlns="http://www.w3.org/1999/xhtml"><p>The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression.</p></abstract>

The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression.

Characterization of Three Generations of Transgenic Pigs Expressing the HLA-E Gene

Department of Biochemistry and Biotechnology

Department of Animal Reproduction, National Research Institute of Animal Production

Department of Biotechnology and Food Microbiology

Institute of Human Genetics, Polish Academy of Sciences

Department of Mathematical and Statistical Methods

Annals of Animal Science

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important...

Characterization of Three Generations of Transgenic Pigs Expressing the HLA-E Gene

Published Online: Nov 02, 2018

Page range: 919 - 935

Received: Mar 08, 2018

Accepted: Jul 13, 2018

DOI: https://doi.org/10.2478/aoas-2018-0034

Keywords
transgenic pigs, transgene expression, xenotransplantation, transgene stability, transgene inheritance

© 2018 Magdalena Hryhorowicz et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Characterization of Three Generations of Transgenic Pigs Expressing the HLA-E Gene

Published Online: Nov 02, 2018

Page range: 919 - 935

Received: Mar 08, 2018

Accepted: Jul 13, 2018

DOI: https://doi.org/10.2478/aoas-2018-0034

Keywordstransgenic pigs, transgene expression, xenotransplantation, transgene stability, transgene inheritance

© 2018 Magdalena Hryhorowicz et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
transgenic pigs, transgene expression, xenotransplantation, transgene stability, transgene inheritance