Browsing by Author "Prather, Randall S."

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    Partial loss of interleukin 2 receptor gamma function in pigs provides mechanistic insights for the study of human immunodeficiency syndrome
    Choi, Yun-Jung; Lee, Kiho; Park, Woo-Jin; Kwon, Deug-Nam; Park, Chankyu; Do, Jeong Tae; Song, Hyuk; Cho, Seong-Keun; Park, Kwang-Wook; Brown, Alana N.; Samuel, Melissa S.; Murphy, Clifton N.; Prather, Randall S.; Kim, Jin-Hoi (Impact Journals, 2016-08-09)
    In this study, we described the phenotype of monoallelic interleukin 2 receptor gamma knockout (mIL2RG+/Δ69-368 KO) pigs. Approximately 80% of mIL2RG+/Δ69-368 KO pigs (8/10) were athymic, whereas 20% (2/10) presented a rudimentary thymus. The body weight of IL2RG+/Δ69-368 KO pigs developed normally. Immunological analysis showed that mIL2RG+/Δ69-368 KO pigs possessed CD25+CD44- or CD25-CD44+ cells, whereas single (CD4 or CD8) or double (CD4/8) positive cells were lacking in mIL2RG+/Δ69-368 KO pigs. CD3+ cells in the thymus of mIL2RG+/Δ69-368 KO pigs contained mainly CD44+ cells and/or CD25+ cells, which included FOXP3+ cells. These observations demonstrated that T cells from mIL2RG+/Δ69-368 KO pigs were able to develop to the DN3 stage, but failed to transition toward the DN4 stage. Whole-transcriptome analysis of thymus and spleen, and subsequent pathway analysis revealed that a subset of genes differentially expressed following the loss of IL2RG might be responsible for both impaired T-cell receptor and cytokine-mediated signalling. However, comparative analysis of two mIL2RG+/Δ69-368 KO pigs revealed little variability in the down- and up-regulated gene sets. In conclusion, mIL2RG+/Δ69-368 KO pigs presented a T-B+NK- SCID phenotype, suggesting that pigs can be used as a valuable and suitable biomedical model for human SCID research.
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    Recombination activating gene-2(null) severe combined immunodeficient pigs and mice engraft human induced pluripotent stem cells differently
    Choi, Yun-Jung; Kim, EunSu; Reza, Abu Musa Md Talimur; Hong, Kwonho; Song, Hyuk; Park, Chankyu; Cho, Seong-Keun; Lee, Kiho; Prather, Randall S.; Kim, Jin-Hoi (2017-09-19)
    This study comparatively investigated the transcriptional, physiological, and phenotypic differences of the immune disorder between severe combined immunodeficient (SCID) mouse and pig models. We discovered that the recombination activating gene-2 (Rag-2) SCID mice, but not RAG-2 SCID pigs, showed intense, infrequent, and mild cluster of CD3(+)-, CD4(+)-, and CD8(+) signals respectively, suggesting that distinct species-specific effects exist. Furthermore, the expression of six relevant genes (NFATC1, CD79B, CD2, BLNK, FOXO1, and CD40) was more downregulated than that in the Rag-2 SCID mice, which provides a partial rationale for the death of T/B cells in the lymphoid organs of RAG-2 SCID pigs but not in Rag-2 SCID mice. Further, NK cell maturation-related gene expression was significantly lower in RAG-2 SCID pigs than in Rag-2 SCID mice. Consistently, the RAG-2 SCID pigs, but not Rag-2 SCID mice, developed human induced pluripotent stem cell-derived teratomas that were the same as those of perforin/Rag-2 SCID mice. Therefore, these unexpected findings indicate the superiority of RAG-2 SCID pigs over Rag-2 SCID mice as a suitable model for investigating human diseases.
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    Use of gene-editing technology to introduce targeted modifications in pigs
    Ryu, Junghyun; Prather, Randall S.; Lee, Kiho (2018-01-29)
    Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer (SCNT) to generate genetically engineered (GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and the Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks (DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining (NHEJ) or homology direct repair (HDR). Random insertions or deletions (indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We will also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.