Fetal bone engraftment reconstitutes the immune system in pigs with severe combined immunodeficiency.

Genetic modification of genes such as recombination activating gene 2 (RAG2) or interleukin-2 receptor-γ (IL2RG) results in pigs exhibiting severe combined immunodeficiency (SCID). Pigs presenting a SCID phenotype are important animal models that can be used to establish xenografts and to study immune system development and various immune-related pathologies. However, due to their immunocompromised nature, SCID pigs have shortened lifespans and are notoriously difficult to maintain. The failure-to-thrive phenotype makes the establishment of a breeding population of RAG2/IL2RG double-knockout pigs virtually impossible. Here, to overcome this limitation, we investigated whether reconstituting the immune system of SCID piglets with a fetal bone allograft would extend their lifespan. Following intramuscular transplantation, allografts gave rise to lymphocytes expressing T cell (CD3, CD4 and CD8), B cell (CD79α) and natural killer cell (CD335) lineage markers, which were detected in circulation as well in the spleen, liver, bone marrow and thymic tissues. The presence of lymphocytes indicates broad engraftment of donor cells in the recipient SCID pigs. Unlike unreconstituted SCID pigs, the engrafted animals thrived and reached puberty under standard housing conditions. This study demonstrates a novel method to extend the survival of SCID pigs, which may improve the availability and use of SCID pigs as a biomedical animal model.

Impaired Skeletal Development by Disruption of Presenilin-1 in Pigs and Generation of Novel Pig Models for Alzheimer's Disease.

Background: Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species. Objective: To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model. Methods: CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction. Results: Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases. Conclusions: PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.

Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs†.

Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9-/-) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9-/- and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9-/-), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9-/- gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs.