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

Abstract

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 Recombination activating gene-1 (Rag-1)-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 Rag-1 gene targeting vectors for the Exon 2 region and obtained heterozygous/homozygous Rag-1 knockout cell colonies using SCNT. We generated two Rag-1-deficient neonatal piglets and compared them with wild-type neonatal piglets. FACS analysis showed that Rag-1 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 Rag-1-deficient piglets. These results confirm that Rag-1 is necessary for the generation of lymphocytes in pigs, and Rag-1-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 Rag-1 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.

SCID pigs: An emerging large animal NK model

“…In an effort to harness this model potential, the SCID condition had been transgenically introduced into mice, rats, and recently swine 12,[32][33][34][35][36][37][38] . The pig offers a large animal model with more similar genetics, anatomy, and physiology to humans.…”

“…Though the SCID phenotype is artificially achievable by a variety of genetic manipulations, swine researchers have focused on targeting IL2RG and/or the RAG 1/2 knockouts 12,[32][33][34][35][36][37] . Engineered IL2RG SCID pigs have been created using serial nuclear transfer 37 , zinc finger nucleases (ZFNs) 12 , or the clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 system 34 technologies.…”

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART−/−IL2RG−/Y SCID Pigs

“…In 2012, we discovered the first naturally occurring SCID pigs (1,2), caused by mutations within the ARTEMIS gene, resulting in a T − B − NK + SCID phenotype (3,4). Since then, pigs with mutations in RAG1 (5,6), RAG2 (7,8), IL2RG (9-11), and RAG2/IL2RG (12) have also been generated through different mutagenic approaches. Within the past few years, such SCID pigs are now being utilized by cancer (13), disease model (12), and stem cell therapy (7) researchers.…”

Organ Fabrication Using Pigs as An in Vivo Bioreactor

“…To achieve this, techniques for suppressing im-munity in the pig to enable acceptance of human cells and for inducing immune tolerance to cells must be evaluated. Immunosuppression has been successful in pigs [7][8][9][10][11] ; however, unlike for small animals, management of the rearing of these immunocompromised pigs makes organ production scientifically and economically difficult. 12 In this review, we first provide an overview of recent studies on the production of human tissues and organs in living pigs.…”