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The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Metabolic disorders are characterized by an imbalance in muscle fiber composition, and a potential therapeutic approach involves increasing the proportion of oxidative muscle fibers. Prokineticin receptor 1 (PROKR1) is a G protein-coupled receptor that plays a role in various metabolic functions, but its specific involvement in oxidative fiber specification is not fully understood. Here, we investigated the functions of PROKR1 in muscle development to address metabolic disorders and muscular diseases. A meta-analysis revealed that the activation of PROKR1 upregulated exercise-responsive genes, particularly nuclear receptor subfamily 4 group A member 2 (NR4A2). Further investigations using ChIP-PCR, luciferase assays, and pharmacological interventions demonstrated that PROKR1 signaling enhanced NR4A2 expression by Gs-mediated phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in both mouse and human myotubes. Genetic and pharmacological interventions showed that the PROKR1-NR4A2 axis promotes the specification of oxidative muscle fibers in both myocytes by promoting mitochondrial biogenesis and metabolic function. Prokr1-deficient mice displayed unfavorable metabolic phenotypes, such as lower lean mass, enlarged muscle fibers, impaired glucose, and insulin tolerance. These mice also exhibited reduced energy expenditure and exercise performance. The deletion of Prokr1 resulted in decreased oxidative muscle fiber composition and reduced activity in the Prokr1-CREB-Nr4a2 pathway, which were restored by AAV-mediated Prokr1 rescue. In summary, our findings highlight the activation of the PROKR1-CREB-NR4A2 axis as a mechanism for increasing the oxidative muscle fiber composition, which positively impacts overall metabolic function. This study lays an important scientific foundation for the development of effective muscular-metabolic therapeutics with unique mechanisms of action., Competing Interests: Competing interests statement:The authors declare no competing interest.

Gene therapy and rebalancing therapy have emerged as promising approaches for treating hemophilia A, but there are limitations, such as temporary efficacy due to individual differences. Genome editing for hemophilia has shown long-term therapeutic potential in preclinical trials. However, a cautious approach is necessary because genome editing is irreversible. Therefore, we attempted to induce low-level human factor 8 (hF8) gene knockin (KI) using 244- cis lipid nanoparticles and low-dose adeno-associated virus to minimize side effects and achieve a therapeutic threshold in hemophilia A mice. We selected the serpin family C member 1, SerpinC1 , locus as a target to enable a combined rebalancing strategy with h F8 KI to augment efficacy. This strategy improved blood coagulation activity and reduced hemophilic complications without adverse effects. Furthermore, hemophilic mice with genome editing exhibit enhanced survival for 40 weeks. Here, we demonstrate an effective, safe, and sustainable treatment for hemophilia A. This study provides valuable information to establish safe and long-term genome-editing-mediated treatment strategies for treating hemophilia and other protein-deficient genetic diseases., Competing Interests: Hyukjin Lee received a research grant from ST Pharm, and ST Pharm also has a license for certain intellectual property invented at Ewha Womans University., (© 2023 The Authors.)

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (h F9 ) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [ Serpinc1 ]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, h F9 coding sequences were integrated into approximately 3% of the target locus. h F9 KI yielded approximately 1,000 ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63 weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and h F9 KI using LNP and AAV., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, APOC3 , in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of APOC3 editing using a small Cas9 protein derived from Campylobacter jejuni (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing in vitro or in vivo . Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient APOC3 -editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases., Competing Interests: H.-K.O., H.R.L., K.J.L., J.L., H.H.L., S.K., and D.W.S. were employed by ToolGen. K.J.L., U.G.K., S.K., and D.W.S. filed patent applications based on this work. The authors declare no competing financial interests., (© 2022 The Authors.)

Natural soluble antagonist and decoy receptor on the surface of the cell membrane are evolving as crucial immune system regulators as these molecules are capable of recognizing, binding, and neutralizing (so-called inhibitors) their targeted ligands. Eventually, these soluble antagonists and decoy receptors terminate signaling by prohibiting ligands from connecting to their receptors on the surface of cell membrane. Interleukin-18 binding protein (IL-18BP) participates in regulating both Th1 and Th2 cytokines. IL-18BP is a soluble neutralizing protein belonging to the immunoglobulin (Ig) superfamily as it harbors a single Ig domain. The Ig domain is essential for its binding to the IL-18 ligand and holds partial homology to the IL-1 receptor 2 (IL-1R2) known as a decoy receptor of IL-1α and IL-1β. IL-18BP was defined as a unique soluble IL-18BP that is distinct from IL-18Rα and IL-18Rβ chain. IL-18BP is encoded by a separated gene, contains 8 exons, and is located at chr.11 q13.4 within the human genome. In this review, we address the difference in the biological activity of IL-18BP isoforms, in the immunity balancing Th1 and Th2 immune response, its critical role in autoimmune diseases, as well as current clinical trials of recombinant IL-18BP (rIL-18BP) or equivalent., Competing Interests: The authors declare no conflict of interest.

IL-32 plays a contradictory role such as tumor proliferation or suppressor in cancer development depending on the cancer type. In most cancers, it was found that the high expression of IL-32 was associated with more proliferative and progression of cancer. However, studying the isoforms of IL-32 cytokine has placed its paradoxical role into a wide range of functions based on its dominant isoform and surrounding environment. IL-32β, for example, was found mostly in different types of cancer and associated with cancer expansion. This observation is legitimate since cancer exhibits some hypoxic environment and IL-32β was known to be induced under hypoxic conditions. However, IL-32θ interacts directly with protein kinase C-δ reducing NF-κB and STAT3 levels to inhibit epithelial-mesenchymal transition (EMT). This effect could explain the different functions of IL-32 isoforms in cancer. However, pro- or antitumor activity which is dependant on obesity, gender, and age as it relates to IL-32 has yet to be studied. Obesity-related IL-32 regulation indicated the role of IL-32 in cancer metabolism and inflammation. IL-32-specific direction in cancer therapy is difficult to conclude. In this review, we address that the paradoxical effect of IL-32 on cancer is attributed to the dominant isoform, cancer type, tumor microenvironment, and genetic background. IL-32 seems to have a contradictory role in cancer. However, investigating multiple IL-32 isoforms could explain this doubt and bring us closer to using them in therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shim, Lee, Hisham, Kim, Nguyen, Taitt, Hwang, Jhun, Park, Lee, Yeom, Kim, Kim and Kim.)

Cytokines are significantly associated with the homeostasis of immune responses in health and disease. Interleukin-32 (IL-32) is a cytokine originally discovered in natural killer cell transcript 4. IL-32 with different disorders has been described in terms of pathogenesis and the progression of diseases. Clinical studies have investigated IL-32 under various conditions, such as viral infection, autoimmune diseases, inflammatory diseases, certain types of cancer, vascular disease, and pulmonary diseases. The high expression of IL-32 was identified in different tissues with various diseases and found to have multiple transcripts of up to seven isoforms. However, the purification and biological activities of these isoforms have not been investigated yet. Therefore, in this study, we purified and compared the biological activity of recombinant IL-32 (rIL-32) isoforms. This is the first time for seven rIL-32 isoforms (α, β, δ, γ, ϵ, ζ, and θ) to be cloned and purified using an Escherichia coli expression system. Next, we evaluate the biological activities of these seven rIL-32 isoforms, which were used to treat different types of cells by assessing the levels of inflammatory cytokine production. The results revealed that rIL-32θ possessed the most dominant biological activity in both immune and non-immune cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shim, Lee, Hisham, Kim, Nguyen, Taitt, Hwang, Jhun, Park, Lee, Yeom, Kim, Kim and Kim.)

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

Laboratory animal medicine (LAM) is a corner stone of animal-based research and has been a veterinary specialty for over 60 y. Today 5 Colleges of LAM (American, European, Japanese, Korean, and Indian) that certify specialists (Diplomates) in LAM are members of the International Association of Colleges of LAM (IACLAM). Goals of IACLAM are to support the development of new Colleges of LAM, to harmonize expectations for the knowledge and skills of newly certified LAM Diplomate, and to harmonize the standards (best practices) for training and examination of candidates among the member Colleges. IACLAM recently conducted an in-depth review and comparison of oversight, training, credentialing, and examination standards in the 5 Colleges as part of an initiative to create a framework for harmonization and consistency for these activities across the 5 Colleges. The process has led to an agreement on recommendations for knowledge and skill requirements for a newly certified Diplomate, as described by each College in a detailed role delineation document (RDD). The RDD is based on task analyses of the work responsibilities of laboratory animal veterinary Diplomates. This agreement is an important step toward the goal of global harmonization of LAM Diplomate training. Further efforts are planned for areas such as training, research, publication, and examination. This paper describes the role and content of the RDD and lists the differences and similarities among the RDDs of 5 Colleges of LAM.

Recently, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (B.1.1.529) Omicron variant originated from South Africa in the middle of November 2021. SARS-CoV-2 is also called coronavirus disease 2019 (COVID-19) since SARS-CoV-2 is the causative agent of COVID-19. Several studies already suggested that the SARS-CoV-2 Omicron variant would be the fastest transmissible variant compared to the previous 10 SARS-CoV-2 variants of concern, interest, and alert. Few clinical studies reported the high transmissibility of the Omicron variant but there is insufficient time to perform actual experiments to prove it, since the spread is so fast. We analyzed the SARS-CoV-2 Omicron variant, which revealed a very high rate of mutation at amino acid residues that interact with angiostatin-converting enzyme 2. The mutation rate of COVID-19 is faster than what we prepared vaccine program, antibody therapy, lockdown, and quarantine against COVID-19 so far. Thus, it is necessary to find better strategies to overcome the current crisis of COVID-19 pandemic., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest., (Copyright © 2021. The Korean Association of Immunologists.)

Charcot-Marie-Tooth disease type 2A (CMT2A) is the most common hereditary axonal neuropathy caused by mutations in MFN2 encoding Mitofusin-2, a multifunctional protein located in the outer mitochondrial membrane. In order to study the effects of a novel MFN2 K357T mutation associated with early onset, autosomal dominant severe CMT2A, we generated a knock-in mouse model. While Mfn2 K357T/K357T mouse pups were postnatally lethal, Mfn2 +/K357T heterozygous mice were asymptomatic and had no histopathological changes in their sciatic nerves up to 10 months of age. However, immunofluorescence analysis of Mfn2 +/K357T mice revealed aberrant mitochondrial clustering in the sciatic nerves from 6 months of age, in optic nerves from 8 months, and in lumbar spinal cord white matter at 10 months, along with microglia activation. Ultrastructural analyses confirmed dysmorphic mitochondrial aggregates in sciatic and optic nerves. After exposure of 6-month-old mice to lipopolysaccharide, Mfn2 +/K357T mice displayed a higher immune response, a more severe motor impairment, and increased CNS inflammation, microglia activation, and macrophage infiltrates. Overall, ubiquitous Mfn2 K357T expression renders the CNS and peripheral nerves of Mfn2 +/K357T mice more susceptible to mitochondrial clustering, and augments their response to inflammation, modeling some cellular mechanisms that may be relevant for the development of neuropathy in patients with CMT2A.

The microrchidia (MORC)-family CW-type zinc finger 2 (MORC2) gene is related to DNA repair, adipogenesis and epigenetic silencing via the human silencing hub (HUSH) complex. MORC2 missense mutation is known to cause peripheral neuropathy of Charcot-Marie-Tooth disease type 2 Z (CMT2Z). However, there have been reports of peripheral and central neuropathy in patients, and the disease has been co-categorized with developmental delay, impaired growth, dysmorphic facies and axonal neuropathy (DIGFAN). The etiology of MORC2 mutation-mediated neuropathy remains uncertain. Here, we established and analyzed Morc2a p.S87L mutant mice. Morc2a p.S87L mice displayed the clinical symptoms expected in human CMT2Z patients, such as axonal neuropathy and skeletal muscle weakness. Notably, we observed severe central neuropathy with cerebella ataxia, cognition disorder and motor neuron degeneration in the spinal cord, and this seemed to be evidence of DIGFAN. Morc2a p.S87L mice exhibited an accumulation of DNA damage in neuronal cells, followed by p53/cytochrome c/caspase 9/caspase 3-mediated apoptosis. This study presents a new mouse model of CMT2Z and DIGFAN with a Morc2a p.S87L mutation. We suggest that neuronal apoptosis is a possible target for therapeutic approach in MORC2 missense mutation. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

Neuronal nitric oxide synthase (nNOS) has various roles as a neurotransmitter. However, studies to date have produced insufficient data to fully support the correlation between nNOS and bowel motility. This study aimed to investigate the correlation between nNOS expression and gastrointestinal (GI) tract motility using a stress-induced neonatal maternal separation (NMS) mouse model. In this study, we generated a genetically modified mouse with the HiBiT sequence knock-in into the nNOS gene using CRISPR/Cas9 for analyzing accurate nNOS expression. nNOS expression was measured in the stomach, small intestine, large intestine, adrenal gland, and hypothalamus tissues after establishing the NMS model. The NMS model exhibited a significant increase in nNOS expression in large intestine, adrenal gland, and hypothalamus. Moreover, a significant positive correlation was observed between whole gastrointestinal transit time and the expression level of nNOS. We reasoned that NMS induced chronic stress and consequent nNOS activation in the hypothalamic-pituitary-adrenal (HPA) axis, and led to an excessive increase in intestinal motility in the lower GI tract. These results demonstrated that HiBiT is a sensitive and valuable tool for analyzing in vivo gene activation, and nNOS could be a biomarker of the HPA axis-linked lower intestinal tract dysfunction.

The global crisis caused by the coronavirus disease 2019 (COVID-19) led to the most significant economic loss and human deaths after World War II. The pathogen causing this disease is a novel virus called the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of December 2020, there have been 80.2 million confirmed patients, and the mortality rate is known as 2.16% globally. A strategy to protect a host from SARS-CoV-2 is by suppressing intracellular viral replication or preventing viral entry. We focused on the spike glycoprotein that is responsible for the entry of SARS-CoV-2 into the host cell. Recently, the US Food and Drug Administration/EU Medicines Agency authorized a vaccine and antibody to treat COVID-19 patients by emergency use approval in the absence of long-term clinical trials. Both commercial and academic efforts to develop preventive and therapeutic agents continue all over the world. In this review, we present a perspective on current reports about the spike glycoprotein of SARS-CoV-2 as a therapeutic target., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest., (Copyright © 2021. The Korean Association of Immunologists.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a positive-sense single-stranded RNA (+ssRNA) that causes coronavirus disease 2019 (COVID-19). The viral genome encodes twelve genes for viral replication and infection. The third open reading frame is the spike ( S ) gene that encodes for the spike glycoprotein interacting with specific cell surface receptor - angiotensin converting enzyme 2 (ACE2) - on the host cell membrane. Most recent studies identified a single point mutation in S gene. A single point mutation in S gene leading to an amino acid substitution at codon 614 from an aspartic acid 614 into glycine (D614G) resulted in greater infectivity compared to the wild type SARS-CoV2. We were interested in investigating the mutation region of S gene of SARS-CoV2 from Korean COVID-19 patients. New mutation sites were found in the critical receptor binding domain (RBD) of S gene, which is adjacent to the aforementioned D614G mutation residue. This specific sequence data demonstrated the active progression of SARS-CoV2 by mutations in the RBD of S gene. The sequence information of new mutations is critical to the development of recombinant SARS-CoV2 spike antigens, which may be required to improve and advance the strategy against a wide range of possible SARS-CoV2 mutations., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest., (Copyright © 2020. The Korean Association of Immunologists.)

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Campylobacter jejuni Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse Tyr or Foxn1 and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted in silico and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner., Competing Interests: JYL, HSB, SJK and OJK are employees of ToolGen Inc, a company develops products based on CRISPR/Cas9 system. SGP is employee of TheragenEtex Bio Inc, a company providing genome sequencing., (© 2020 The Authors.)

Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings will provide insights in the improved performance of engineered nucleases when they are applied in vivo.

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras , Trp53 , Ink4a , Smad4 , and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Korean Society of Veterinary Science.)

The oxidation of guanine to 8-oxoguanine (8-oxoG) is the most common type of oxidative DNA lesion. There is a growing body of evidence indicating that 8-oxoG is not only pre-mutagenic, but also plays an essential role in modulating gene expression along with its cognate repair proteins. In this study, we investigated the relationship between 8-oxoG formed under intrinsic oxidative stress conditions and gene expression in adipose and lung tissues of juvenile mice. We observed that transcriptional activity and the number of active genes were significantly correlated with the distribution of 8-oxoG in gene promoter regions, as determined by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), and 8-oxoG and RNA sequencing. Gene regulation by 8-oxoG was not associated with the degree of 8-oxoG formation. Instead, genes with GC-rich transcription factor binding sites in their promoters became more active with increasing 8-oxoG abundance as also demonstrated by specificity protein 1 (Sp1)- and estrogen response element (ERE)-luciferase assays in human embryonic kidney (HEK293T) cells. These results indicate that the occurrence of 8-oxoG in GC-rich Sp1 binding sites is important for gene regulation during adipose tissue development.

Albumin is a promising candidate as a biomarker for potential disease diagnostics and has been extensively used as a drug delivery carrier for decades. In these two directions, many albumin-detecting probes and exogenous albumin-based nanocomposite delivery systems have been developed. However, there are only a few cases demonstrating the specific interactions of exogenous probes with albumin in vivo, and nanocomposite delivery systems usually suffer from tedious fabrication processes and potential toxicity of the complexes. Herein, we demonstrate a facile "one-for-all" switchable nanotheranostic (NanoPcS) for both albumin detection and cancer treatment. In particular, the in vivo specific binding between albumin and PcS, arising from the disassembly of injected NanoPcS, is confirmed using an inducible transgenic mouse system. Fluorescence imaging and antitumor tests on different tumor models suggest that NanoPcS has superior tumor-targeting ability and the potential for time-modulated, activatable photodynamic therapy.

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.

Background: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder involving changes in normal bowel movements. The pathophysiology of IBS is not clearly understood owing to the lack of identifiable pathological abnormalities and reliable biomarkers., Aim: The aim of this study was to discover the novel and reliable biomarker for IBS., Method: In this study, neonatal maternal separation (NMS) stress model was used for the IBS mouse model. Further assessment was conducted with whole gastrointestinal transit test, quantitative RT-PCR, histological examination, and western blot., Results: Male pups developed symptoms similar to those of human IBS with diarrhea (IBS-D), such as low-grade inflammation, stool irregularity, and increased bowel motility. NMS stress influenced to the interstitial cells of Cajal (ICC) and induced altered bowel motility, resulting in IBS-D-like symptoms. In addition, we found neuronal nitric oxide synthase (nNOS) to be a novel biomarker for ICC under NMS stress. nNOS expression was only observed in the ICC of the submucosal plexus of IBS-D mice, and the inhibition of nNOS changed the phenotype from IBS-D to IBS with constipation., Conclusion: Our study demonstrates that early-life stress can influence to ICC and modulate bowel activity and that nNOS might be used as a biomarker for ICC stimulation in IBS.

Alopecia areata (AA) is an autoimmune disease that results in spot baldness in humans. Adequate animal models for AA are currently lacking. The objective of this study was to elucidate the mechanism of autoimmune-like alopecia (ALA) in C57BL/6.CD80CD86-deficient (B6.CD80CD86 -/- ) mice. Incidence and severity of alopecia were analysed in 58 B6.CD80CD86 -/- mice using histological examination, flow cytometry, multiplex enzyme-linked immunosorbent assay, quantitative RT-PCR and CD25 inhibition test. Both male and female B6.CD80CD86 -/- mice showed almost 100% incidence of hair loss at 40 weeks of age. Moreover, CD4+FoxP3+Treg (Treg) cell population in B6.CD80CD86 -/- mice was significantly lower than in B6 mice, which presumably underlined autoimmune reaction. Histologically, B6.CD80CD86 -/- mice showed CD4+ and CD8+ T-cell infiltration around terminal follicle region and exhibited hair follicle destruction in the anagen or catagen stage. Negative correlation between the number of CD4+FoxP3+ Tregs and ALA was confirmed by the CD25 depletion test in B6 mice, as follicle destruction was similar to that observed in B6.CD80CD86 -/- animals. CD80CD86 deficiency disrupted CD4+FoxP3+ Treg homoeostasis and prompted the development of ALA. We demonstrated that B6.CD80CD86 -/- mice might have several advantages as an ALA model, because they exhibited high incidence of disease phenotype and epipathogenesis similar to that observed in human AA., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Anagen effluvium develops because of disturbances in the hair follicle cycle, leading to acute and severe hair loss in humans. The objective of this study was to establish a mouse model of anagen effluvium by 5-bromo-2'-deoxyuridine (BrdU) treatment, and evaluate the pathological changes and underlying mechanisms. We treated 9-10-day-old pups and 3-7-week-old C57BL/6 mice with BrdU. After successfully inducing hair loss in the neonatal pups, microscopic, immunohistochemical and flow cytometry analyses were conducted. BrdU induced early onset alopecia in neonates and caused epidermal thickening and hair shaft breakage. BrdU appeared to incorporate the CD326-positive keratinocyte layer and induced p53-related apoptosis. Keratinocyte apoptosis caused immune cell infiltration in the dermal region; M2 macrophages and neutrophils were dominant. The BrdU-induced hair loss was dose-dependent, and alopecia was visible at a dose range of 25-200 μg/g bodyweight. The BrdU-induced anagen effluvium mouse model is novel and easily established by administrating four simple BrdU injections to pups; these mice showed synchronized onset of alopecia symptoms with little individual variation. Moreover, this model showed an alopecia phenotype similar to that of human anagen effluvium with acute, severe and widespread hair loss., (© 2016 Japanese Dermatological Association.)

Some G-protein-coupled receptors have been reported to require accessory proteins with specificity for proper functional expression. In this study, we found that CXCR1 interacted with REEP5 and REEP6, but CXCR2 did not. Overexpression of REEP5 and REEP6 enhanced IL-8-stimulated cellular responses through CXCR1, whereas depletion of the proteins led to the downregulation of the responses. Although REEPs enhanced the expression of a subset of GPCRs, in the absence of REEP5 and REEP6, CXCR1 was expressed in the plasma membrane, but receptor internalization and intracellular clustering of β-arrestin2 following IL-8 treatment were impaired, suggesting that REEP5 and REEP6 might be involved in the ligand-stimulated endocytosis of CXCR1 rather than membrane expression, which resulted in strong cellular responses. In A549 lung cancer cells, which endogenously express CXCR1, the depletion of REEP5 and REEP6 significantly reduced growth and invasion by downregulating IL-8-stimulated ERK phosphorylation, actin polymerization and the expression of genes related to metastasis. Furthermore, an in vivo xenograft model showed that proliferation and metastasis of A549 cells lacking REEP5 and REEP6 were markedly decreased compared to the control group. Thus, REEP5 and REEP6 could be novel regulators of G-protein-coupled receptor signaling whose functional mechanisms differ from other accessory proteins.

Recently, we found that maternal stress could induce premature mammary gland involution in interleukin 10 knock out (IL-10 -/- ) mice. To elucidate correlation between stress, IL-10, and mammary gland involution, corticosterone was injected into the lactating wild type and IL-10-deficient mice and assessed mammary gland phenotype. Repetitive corticosterone injection developed premature mammary gland involution only in B6.IL-10 -/- mice; moreover, it induced alopecia in nursing pups. Corticosterone injection induced several typical changes such as mammary gland epithelial cell apoptosis, macrophage infiltration, fat deposition in adipocyte, STAT3 phosphorylation, and upregulation of tyrosine hydroxylase gene in adrenal gland. Overall incidence of pup alopecia and mammary gland involution was relatively high in corticosterone than control B6.IL-10 -/- group (57% vs. 20%). Our finding demonstrates that IL-10 is important for stress modulation, and B6.Il-10 -/- with corticosterone has several advantage such as simple to establish, well-defined onset of mammary gland involution, high incidence, and inducing pup alopecia.

In the present study we used an empty zona pellucida derived from hatched blastocysts as an alternative source for embryo aggregation and compared results with the conventional microwell method. Denuded 4-cell stage porcine embryos were aggregated by introduction into an empty zona or placement within a concave microwell. The present study showed that although the rate of aggregate formation was similar, the blastocyst rates and allocation of more cells to the inner cell mass (ICM) in the resultant aggregates were increased significantly more in the empty zona than in the microwell. Notably, using an empty zona showed no limitations with regards to the increased number of embryos aggregated or embryonic stages for aggregation, while partial or no aggregation frequently occurred in the microwell. The discrepancy may be due to the difference of microenvironments where the embryos were placed namely, the presence/absence of zona pellucida. We hypothesize the success of the empty zona in generating aggregates is due to the physical aggregation of individual embryos allowing closer contact between the blastomeres and/or embryos compared with a concave microwell. These results indicate that aggregation conditions could influence overall production efficiency and developmental potential of aggregates, suggesting physical restraint via empty zona that provide three-dimensional pressures is an important factor for successful embryo aggregation.

Tiglium seed is a seed of mature Croton Tiglium Linne containing croton oils, which have been traditionally used as laxative or purgative. As it contains phorbol derivatives, we investigated the mutagenicity and tumor-promoting activity of Tiglium seed. Tiglium seed extract produced the mutagenic responses in five Salmonella typhimurium strains in Ames assay, whereas it did not alter the frequencies of chromosomal aberrations or micronuclei, indicating that it exerted the mutagenic potential, not clastogenicity. Accompanied with phosphorylation of connexin43 (Cx43) and extracellular signal-regulated kinases 1/2 (ERK1/2), Tiglium seed extract inhibited gap junctional intercellular communication (GJIC) associated with tumor-promoting potential. Importantly, these effects were blocked by a protein kinase C (PKC) inhibitor or mitogen-activated protein kinase (MAPKs) inhibitors, suggesting that Tiglium seed-induced GJIC inhibition was regulated by phosphorylation of Cx43 via PKC and MAPKs signaling. In conclusion, Tiglium seed has mutagenicity, possibly linking to tumor-promoting potential through the dysfunction of GJIC.

Organ transplantation is limited by the shortage of human organs. Many studies have sought to overcome this hurdle by using animal organs. Porcine organs, especially from miniature pigs, have been used for organ xenotransplantation rather than nonhuman primates. While the molecular profiling for transplantation is well known in humans and rodents, the situation for pigs is almost completely unknown. The present study examined protein regulation of the developing stages of the pancreatic proteome (4 day-old miniature neonate, 19 day-old miniature piglet, and 14 month-old miniature adult pigs) using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry. Thirteen different expressed spots were observed and nine were identified. The data presented within this study provides critical direction relating to the development of pancreas of miniature pigs, which will assist future proteome analysis of the pancreas, and advance our understanding of the hurdles facing xenotransplantation.

Background: The interleukin 10 deficient mice (IL-10(-/-)) showed high incidence of pup alopecia compared to other strains, and pup alopecia was caused by skin inflammation and was recoverable. Pup alopecia of B6.IL-10(-/-) might be related with maternal factor and interleukin-10 deficient phenotype., Objective: The objectives of this study were elucidating of maternal factors for inflammatory milk production and characterization of pup alopecia in IL-10(-/-) mice., Methods: Incidences of pup alopecia were analyzed with 13 breeding cases. Comparison between control and alopecia pups and its dams, were conducted with histological examination (H&E, TUNEL assay, immunohistochemistry for F4/80, iNOS, CD206, Gr-1, CD4, CD8, CD11c and CD326), fostering test, forced weaning test, qPCR for tyrosine hydroxylase, flow cytometry, IL-10 inhibition test, BMDM stimulation test and LC/MS analysis., Results: Presence of pregnancy in postpartum estrus showed significant correlation with inflammatory milk production and mammary gland involution in B6.IL-10(-/-) mice. There were no different mass in inflammatory milk, but different ionization intensity was detected. Inflammatory milk directly induced hepatocyte steatosis, catagen stage specific hair breaking and alopeicia in pups. Histologically, hypertropy of outer root sheath and macrophage/neutrophil infiltration were typical., Conclusion: B6.IL-10(-/-) dam with stress such as PPE could produce untimely mammary gland involution and inflammatory milk production. Interleukin 10 is important for maternal stress regulation and protecting inflammatory milk production, also influence severity of pup skin inflammation and alopecia. Remarkably, inflammatory milk induced hepatocyte steatosis, and it could indicate there is abnormal lipid metabolism. This was first report for catagen specific alopecia in mouse., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Due to the shortage of human organ donors for transplant, various studies of xenotransplantation, or the use of animal organs instead of human organs, have been carried out. The organs of porcine are thought to be safer and of a more suitable size for xenotransplantationthan those of nonhuman primates. Understanding the levels of expression of proteins, and their post-translational regulation, would be very practical between different species and among developing stages, though the molecular profiling for xenotransplantation has been rarely studied for porcine, while that of human and rodent is well known. Here, in this present study, we report protein regulation of the developing stages of liver (4-day old neonate, 19-day old piglet and 14-month old adult miniature pigs) using 2-DE and MALDI-TOF. From images of the three different stages, a total of 8 spotswhich were differently regulated were identified, and 5 spots were identified with MALDI-TOF MS. The data presented within this study provides critical direction relating to the development of livers of miniature pigs, which will assist future proteome analysis of the liver, and advance our understanding of the hurdles facing xenotransplantaion.

This study was performed to determine the influence of pig breed and gender on the ultimate pH and physicochemical properties of pork. The correlations between pH and pork quality traits directly related to carcass grade, and consumer's preference were also evaluated. The pH and meat grading scores for cold carcasses of 215 purebred pigs (Duroc, Landrace, and Yorkshire) from four different farms were obtained. Meat quality parameters of the pork loin were analyzed. Duroc and female animals were more affected compared to other breeds and male pigs. Duroc animals had the highest ultimate pH, carcass back fat thickness, marbling scores, yellowness, and fat content (p<0.05). Landrace pigs had the highest color lightness and cooking loss values (p<0.05). Among all trait parameters, marbling scores showed the highest significant differences when evaluating the impact of breed and gender on meat quality characteristics (p<0.001). Ultimate pH was positively correlated with carcass weight (0.20), back fat thickness (0.19), marbling score (0.17), and color score (0.16) while negatively correlated with cooking loss (-0.24) and shear force (-0.20). Therefore, pork samples with lower ultimate pH had lower cooking loss, higher lightness, and higher shear force values irrespective of breed.

We analyzed 72 items related to serum biochemistry and hematology in 85 specific pathogen-free (SPF) Seoul National University (SNU) miniature pigs aged 1- to 36-months which originated from a Minnesota miniature pig. Almost all examined items were similar between male and female pigs. However, some items such as Cr level, B/C ratio, C.R.F, LDH, LAP and T4 were significantly different between male and female pigs (P<0.05). Thirty four examined items showed age-related changes, and the significant changes were observed in animals less than six months old. The values for BUN, K, uric acid, Ca, Ca(++), and Pi were significantly higher in pigs younger than six months of age, which might reflect poor kidney function in young pigs. Additionally, TIBC, UIBC and RDW were significantly higher in young pigs, and RBC, Hb, HCT, MCHC and MCV were significantly lower in young pigs, thus indicating a similar physiology of iron deficiency anemia. These age-related specific phenotypes seemed to be normal, but it should be considered in the long-term experiment using the young pigs. In conclusion, in this study, we defined the normal reference intervals for SPF SNU miniature pigs, and we also determined that there are some physiological differences between the pig genders and ages. This study provides fundamental data for use in experiments involving SPF SNU miniature pigs.

Background and Purpose: Lipopolysaccharide (LPS) induces intestinal dysmotility by alteration of smooth muscle and enteric neuronal activities. However, there is no report on the modulatory effects of LPS on the interstitial cells of Cajal (ICCs). We investigated the effect of LPS and its signal transduction in ICCs., Methods: We performed whole-cell patch clamp and RT-PCR in cultured ICCs from mouse small intestine., Results: LPS suppressed the generation of pacemaker currents of ICCs. The mRNA transcripts for Toll-like receptor 4 (TLR4) were expressed in ICCs. However, the inhibitory action of LPS on pacemaker currents from TLR4(+/+) mice was not present in TLR4(-/-) mice. The inhibitory effects of LPS on ICCs were blocked by glibenclamide (an inhibitor of ATP-sensitive K(+) channels), NS-398 (a COX-2 inhibitor), AH6808 [a prostaglandin E(2) (PGE(2))-EP(2) receptor antagonist], ODQ (an inhibitor of guanylate cyclase) and L-NAME [an inhibitor of nitric oxide synthase (NOS)]. Furthermore, genistein and herbimycin A (tyrosine kinase inhibitors) blocked the LPS-induced inhibitory action on pacemaker activity in ICCs., Conclusions: LPS can activate ICCs to release NO and PGE(2) through TLR4 activation. The released NO and PGE(2) inhibit pacemaker currents by activating ATP-sensitive K(+) channels. The LPS actions are mediated by tyrosine kinase signaling pathways., (Copyright © 2012 S. Karger AG, Basel.)

Background: In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen-free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs., Methods: Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP-grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics., Results: Proportion of islets larger than 200 μm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 μm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue-free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal., Conclusions: Use of the newer bovine nervous tissue-free enzymes including a GMP-grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high-quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered., (© 2011 John Wiley & Sons A/S.)

In an attempt to enable comprehensive high-resolution genotyping of the swine leukocyte antigen (SLA) gene, we performed a systemic analysis of nucleotide polymorphisms at introns 1 and 2 and exon 2 from diverse alleles of SLA-DRB1 and DRB1 pseudogenes. We amplified and cloned 16 partial sequences of SLA-DRB1 and DRB2 introns 1 and 2 from different alleles, and analyzed them together with sequences of four reported SLA-DRB pseudogenes, DRB2, 3, 4, and 5. The results showed the presence of extreme nucleotide variations within introns 1 and 2 of SLA-DRB-related genes including substitutions and deletions. On the basis of these results, we developed a comprehensive genotyping method for SLA-DRB1 by genomic polymerase chain reaction (PCR) and subsequent direct sequencing. A total of 415 animals were genotyped and 67 allelic combinations from 18 DRB1 alleles were identified. Among them, two alleles, SLA-DRB1*kn04 and *kn05, were previously unreported. SLA-DRB1 genotyping results from this study combined with those of SLA-DQB1 from our previous study presented 10 SLA class II haplotypes, three of which were previously unreported. Population analysis using seven different pig breeds showed differences in the allele frequency of SLA-DRB1 among breeds. Our results should benefit biological experiments requiring sequence-level genotyping results of SLA-DRB1 and further study of the complete genetic diversity of SLA-DRB1 using field samples., (© 2011 John Wiley & Sons A/S.)

Seoul National University (SNU) miniature pigs represent a closed colony with 24 founder pigs and a well preserved pedigree. Characterization using mRNA sequence analysis was conducted for 6 swine leukocyte antigen (SLA) loci in parental or founder pigs, and 17 defined alleles were detected. Based on these complete coding sequences, 17 sequence specific primers (SSPs) were designed for polymorphic sites. To validate the specificity of each allele SSP, the PCR-SSP was conducted with defined allele clones as templates. PCR-SSP was conducted with the hot start polymerase and touch-down PCR. The parental or found SNU miniature pigs showed overall SLA class I and II heterozygotes. Using the established PCR-SSP method, we conducted SLA typing for breeding stock including 2 pedigreed pigs and identified the novel SLA class II homozygote haplotye (DRA*0201, DRB1*0403, DQA*0102 and DQB1*0701) and 2 SLA homozygote pig lines: SLA class I Hp-3.0 and class II Hp-0.3, and SLA class I Hp-2.0 and class II Hp-0.2. We thought that our PCR-SSP SLA typing method could be applicable for new SLA homozygote line establishment by assignment and scheduled breeding.

Seoul National University (SNU) miniature pigs are originated from the Minnesota miniature pig. This study was conducted to investigate the maternal origin of SNU (Minnesota) miniature pigs and their phylogenetic relationships by analyzing the mitochondrial DNA (mtDNA) D-loop (control region) sequence. Two mtDNA D-loop sequences of the SNU miniature pigs were identified. On an unweighted pair-group method with an arithmetic mean (UPGMA) phylogenetic tree analysis, the large white was the pig breed closest to the SNU miniature pig, and the pairwise distance analysis showed the same result. While mtDNA sequences of 4 pig breeds which were used to establish Minnesota miniature pig were not known, our result might be different from the history of the Minnesota miniature pig development. In conclusion, we thought that some haplotypes of the Minnesota miniature pig maternally were originated from the Large white pig, or that wild pigs had similar mtDNA sequences to the Large white pig, and all SNU miniature pigs were derived from this colony.

Prediction of islet yield and posttransplant outcome is essential for clinical porcine islet xenotransplantation. Although several histomorphometric parameters of biopsied porcine pancreases are predictive of islet yield, their role in the prediction of in vivo islet potency is unknown. We investigated which histomorphometrical parameter best predicts islet yield and function, and determined whether it enhanced the predictive value of in vitro islet function tests for the prediction of posttransplant outcome. We analyzed the histomorphometry of pancreases from which 60 adult pig islet isolations were obtained. Islet function was assessed using the beta-cell viability index based on flow cytometry analysis, oxygen consumption rate, ADP/ATP ratio, and/or concurrent transplantation into NOD/SCID mice. Receiver operating characteristic (ROC) analysis revealed that only islet equivalent (IEQ)/cm(2) and the number of islets >200 microm in diameter significantly predicted an islet yield of >2000 IEQ/g (p < 0.001 for both) and in vivo islet potency (p = 0.024 and p = 0.019, respectively). Although not predictive of islet yield, a high proportion of large islets (>100 microm in diameter) best predicted diabetes reversal (p = 0.001). Multiple regression analysis revealed that the beta-cell viability index (p = 0.003) and the proportion of islets >100 microm in diameter (p = 0.048) independently predicted mean posttransplant blood glucose level (BGL). When BGL was estimated using both these parameters [area under the ROC curve (AUC), 0.868; 95% confidence interval (CI), 0.730-1.006], it predicted posttransplant outcome more accurately than the beta-cell viability index alone (AUC, 0.742; 95% CI, 0.544-0.939). In conclusion, we identified the best histomorphometric predictors of islet yield and posttransplant outcome. This further enhanced the predictive value of the flow cytometry analysis. These parameters should be useful for predicting islet yield and in vivo potency before clinical adult porcine islet xenotransplantation.

We investigated the prevalence of Helicobacter hepaticus, murine norovirus (MNV), and Pneumocystis carinii and the efficacy of cross-fostering for their eradication in 49 genetically engineered mouse (GEM) strains at our institute. Prevalences of H. hepaticus, MNV, and P. carinii were 33.9, 36.5, and 8.6%, respectively, and immunodeficient strains showed relatively higher prevalence of the 3 pathogens than immunocompetent strains. Additionally, the same immune phenotype strains showed similar prevalences. Furthermore, it was found that NKT cells might play a role in H. hepaticus resistance. Interestingly, there was a high incidence of H. hepaticus and MNV multiple infection. Strains with single or multiple infections of H. hepaticus, MNV, and/or P. carinii were selected, and cross-fostering was conducted. Cross-fosterings were successful at eradicating P. carinii, but there were some failures for H. hepaticus and MNV, and the efficacy of eradication was relatively low compared with previous studies. We thought that this low efficacy might have been due to persistent infection and the high suscepibility to H. hepaticus and MNV of immunodeficient GEM strains. Therefore, cross-fostering may be appropriate for P. carinii eradication, but be inappropriate for repopulation of a new breeding colony with H. hepaticus or MNV infected GEM strains. Our findings provide basic data on maintenance, strain susceptibility, and successful rederivation, especially for GEMs.

Pig blood group antigens may be present on grafted tissue as 16 isoforms, including the major one, A substrance. Seoul National University (SNU) miniature pigs and domestic pigs were used in this study. Polymerase chain reaction (PCR) was performed for the erythrocyte antigen A (EAA) gene, and reverse transcriptase-PCR for pig A transferase fucosyl transferase (FUT) 1 and FUT-2. The hemagglutination test was performed with murine monoclonal anti-A and anti-B antibodies (mAb), and immunohistochemistry with anti-human blood antigen mAb. SNU miniature and domestic pigs showed blood groups A and O. Blood group A SNU miniature pigs expressed either EAA(AA) or EAA(AO) and either S(SS) or S(SO); blood group O miniature pigs expressed EAA(OO) and S(SS) or S(SO), and there was no A(weak). Additionally, blood group A could be divided into blood group A(clotting) and blood group A(not clotting) in hemagglutination tests. Pig A substance was expressed in the lung and kidney in blood group A pigs, but we could not detect pig A substance expression in the lung, kidney, and heart of blood group O pigs or the heart of blood group A pigs. In conclusion, we suggest that blood typing of SNU miniature pigs can be easily performed using immunohistochemistry, PCR, and/or RT-PCR. Molecular-based AO typing described in this study may be useful to select SNU miniature pigs bearing a specific blood group.

Background: Islet cell transplantation is a novel therapeutic modality for the cure of diabetes. Pig islet cells are an attractive substitute for human islet cells; however, they are known to be particularly difficult to isolate because of a weak islet capsule and a tendency to be fragmented during enzymatic digestion. Therefore, parameters favoring successful pig islet isolation were investigated using specific-pathogen-free (SPF) miniature pigs., Methods: Sixty-eight SPF miniature pigs were used for islet isolation. Birth weight, body weight, age, sex, pregnancy history, and the fasting blood glucose levels of each pig were determined. Each pig's general condition was assessed with regard to feeding status and physical activity. Pancreas procurement was performed by one surgical team. Anesthesia duration, operation duration, procedure quality, and perfusate type were recorded. After pancreatectomy, a biopsy was performed for islet density analysis. Decapsulation, cannulation duration, degree of distension, and cold ischemic time were assessed. During islet isolation, pancreas weight, digestion time, and digested tissue proportion were recorded. Isolation results were evaluated by total islet equivalents (IEQ), islet equivalents per gram of pancreas (IEQ/g), isolation index, islet recovery rate, purity, and visual grade. To identify the predictors of higher islet isolation yield, we performed binary logistic regression analysis with significant (P < 0.05) variables from the univariate analysis., Results: The pigs were categorized into high (n = 34) and low yield (n = 34) groups according to the median IEQ/g or total IEQ values. Body weight and age were significantly different between the two groups. Being male or a positive history of pregnancy in females was factors favoring successful islet isolation. General condition assessments failed to estimate islet isolation results. Long anesthesia duration, which might have caused ischemic injury to the pancreas, negatively affected islet isolation results. Decapsulation, cannulation duration, and subsequent pancreas distension were significantly important in successful islet isolation. Inter-lot variability of Liberase was not observed because of screening processes performed before purchase. Isolation index and islet recovery rate correlated well with islet yields., Conclusions: Multivariate analysis using total IEQ and IEQ/g as outcome variables indicated that age older than 2, being male and moderate distension by Liberase injection are major determinants influencing successful islet isolation.