RESUMEN
Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)-/- genetic background. We confirmed ART-/-IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART-/-IL2RG-/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.
Asunto(s)
Trasplante de Médula Ósea , Subunidad gamma Común de Receptores de Interleucina/genética , Inmunodeficiencia Combinada Grave/genética , Animales , Animales Modificados Genéticamente , Antígenos CD34 , Sistemas CRISPR-Cas , Diferenciación Celular , Quimera , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Marcación de Gen , Ingeniería Genética , Supervivencia de Injerto , Reacción Huésped-Injerto , Humanos , Células Asesinas Naturales , Modelos Animales , Porcinos , Linfocitos T/metabolismo , Trasplante HeterólogoRESUMEN
The mean fluorescent intensity obtained using single antigen beads does not represent the titer of the serum sample very well because the degree of saturation of the antigen on the bead varies by individual bead and the human leukocyte antigen antibody-binding site on the bead may not duplicate the activity of the serum's antibodies in vivo. The flow crossmatch appears to come closer to correlating with the titer. As we have shown, the titer will roughly correlate with the flow crossmatch. This is consonant with the fact that the fluorescence level of the crossmatch bears a semi-quantitative relationship to the amount of antibody on the donor cell. The fact that successful transplants can be carried out when the median channel shift of the crossmatch is as high as twice the cutoff suggests that using the mean plus twice the standard deviation of the surrogate negative control may result in too many false positives.