Generation of Recombination Activating Gene-1-Deficient Neonatal Piglets: A Model of T and B Cell Deficient Severe Combined Immune Deficiency

<div><p>Although severe combined immune deficiency (SCID) is a very important research model for mice and SCID mice are widely used, there are only few reports describing the SCID pig models. Therefore, additional research in this area is needed. In this study, we describe the generation of <i>Recombination activating gene-1</i> (<i>Rag-1</i>)-deficient neonatal piglets in Duroc breed using somatic cell nuclear transfer (SCNT) with gene targeting and analysis using fluorescence-activated cell sorting (FACS) and histology. We constructed porcine <i>Rag-1</i> gene targeting vectors for the Exon 2 region and obtained heterozygous/homozygous <i>Rag-1</i> knockout cell colonies using SCNT. We generated two <i>Rag-1</i>-deficient neonatal piglets and compared them with wild-type neonatal piglets. FACS analysis showed that <i>Rag-1</i> disruption causes a lack of Immunoglobulin M-positive B cells and CD3-positive T cells in peripheral blood mononuclear cells. Consistent with FACS analysis, histological analysis revealed structural defects and an absence of mature lymphocytes in the spleen, mesenteric lymph node (MLNs), and thymus in <i>Rag-1</i>-deficient piglets. These results confirm that <i>Rag-1</i> is necessary for the generation of lymphocytes in pigs, and <i>Rag-1</i>-deficient piglets exhibit a T and B cell deficient SCID (T-B-SCID) phenotype similar to that of rodents and humans. The T-B-SCID pigs with <i>Rag-1</i> deficiency generated in this study could be a suitably versatile model for laboratory, translational, and biomedical research, including the development of a humanized model and assessment of pluripotent stem cells.</p>