Browsing by Author "Reyes, Luz M."

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    Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease
    (American Association of Immunologists, 2014-12-01) Reyes, Luz M.; Estrada, Jose L; Wang, Zheng Yu; Blosser, Rachel J.; Smith, Rashod F.; Sidner, Richard A.; Paris, Leela L.; Blankenship, Ross L.; Ray, Caitlin N.; Miner, Aaron C.; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
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    Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein
    (Wiley, 2015-05) Paris, Leela L.; Estrada, Jose L.; Li, Ping; Blankenship, Ross L.; Sidner, Richard A.; Reyes, Luz M.; Montgomery, Jessica B.; Burlak, Christopher; Butler, James R.; Downey, Susan M.; Wang, Zheng-Yu; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    BACKGROUND: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia. METHODS: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers. RESULTS: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion. CONCLUSIONS: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
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    Swine Leukocyte Antigen Class II Is a Xenoantigen
    (Wolters Kluwer, 2018-02) Ladowski, Joseph M.; Reyes, Luz M.; Martens, Gregory R.; Butler, James R.; Wang, Zheng-Yu; Eckhoff, Devin E.; Tector, Matt; Tector, A. Joseph; Surgery, School of Medicine
    BACKGROUND: Over 130 000 patients in the United States alone need a lifesaving organ transplant. Genetically modified porcine organs could resolve the donor organ shortage, but human xenoreactive antibodies destroy pig cells and are the major barrier to clinical application of xenotransplantation. The objective of this study was to determine whether waitlisted patients possess preformed antibodies to swine leukocyte antigen (SLA) class II, homologs of the class II HLA. METHODS: Sera from people currently awaiting solid organ transplant were tested for IgG binding to class II SLA proteins when expressed on mammalian cells. Pig fibroblasts were made positive by transfection with the class II transactivator. As a second expression system, transgenes encoding the alpha and beta chains of class II SLA were transfected into human embryonic kidney cells. RESULTS: Human sera containing IgG specific for class II HLA molecules exhibited greater binding to class II SLA positive cells than to SLA negative cells. Sera lacking antibodies against class II HLA showed no change in binding regardless of the presence of class II SLA. These antibodies could recognize either SLA-DR or SLA-DQ complexes. CONCLUSIONS: Class II SLA proteins may behave as xenoantigens for people with humoral immunity toward class II HLA molecules.