Recent Advance in Genome Editing-Based Gene Modification in Pigs

Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, in vitro electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transfer-derived embryos that have been reconstituted with normal nuclei

Simultaneous Evaluation of Three-Dimensional Lip Kinetics and Tongue Pressure during Swallowing

Objectives: The purpose of this study was to evaluate the amount of lip movement and simultaneous tongue pressure changes on an artificial palatal plate during swallowing. Methods: Subjects were 9 healthy males (25.4 ± 2.1 years of age). Three-dimensional lip movement was measured by a wireless optoelectronic system, and tongue pressure was simultaneously recorded by a sensor sheet attached to the incisive papilla of an artificial palatal plate. Reflective markers were attached to the right and left corners of the mouth to measure the distance between them. All subjects were instructed to swallow 5 mL and 20 mL samples of water at will. The maximum change of distance between the corners of mouth, the maximum tongue pressure, and the time interval between the two maxima (lip-tongue interval) were calculated. Wilcoxon’s test was used to detect significant differences in these measurements between the two volumes. Results: Maximum tongue pressure did not differ significantly between swallowed volumes. The maximum change of distance between the corners of mouth was larger and the lip-tongue interval was significantly shorter with the larger volume. Conclusions: We suggest that swallowing a larger volume is accomplished by larger lip movement rather than larger tongue movement. These results indicate that lip movement during swallowing can be evaluated objectively

Relationships between nasal resistance, adenoids, tonsils, and tongue posture and maxillofacial form in Class II and Class III children

Introduction: The purpose of this study was to clarify the relationships between upper airway factors (nasal resistance, adenoids, tonsils and tongue posture) and maxillofacial form in Class II and III children Methods: Sixty-four subjects (mean age, 9.3 years) with malocclusion were divided into Class II and Class III groups by ANB angle. Nasal resistance was calculated using computational fluid dynamics from cone-beam computed tomography (CBCT) data. Adenoids, tonsils and tongue posture were evaluated in CBCT images. The groups were compared using Mann-Whitney U-tests and Student t-tests. Spearman’s rank test assessed the relationships between upper airway factors and maxillofacial form. Results: Nasal resistance of the Class II group was significantly larger than that of the Class III group (P = 0.005). Nasal resistance of the Class II group was significantly correlated with inferior tongue posture (P < 0.001), and negatively correlated with intermolar width (P = 0.028). Tonsil size of the Class III group was significantly correlated with anterior tongue posture (P < 0.001) and mandibular incisor anterior position (P = 0.007). Anterior tongue posture of the Class III group was significantly correlated with mandibular protrusion. Conclusions: The relationships of upper airway factors differed between Class II and Class III children

Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes

Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding alpha-1,3-galactosyltransferase (GGTA1), which synthesizes the alpha-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B-4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection. (C) 2017 Elsevier Inc. All rights reserved.ArticleTHERIOGENOLOGY.108:29-38(2018)journal articl

Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors

We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100–120 (passages 15–20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the OCT4 and NANOG promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells

HERBST EFFECTS ON PHARYNGEAL AIRWAY VENTILATION

Objective: To evaluate the effect of a Herbst appliance on ventilation of the pharyngeal airway (PA) using computational fluid dynamics (CFD). Materials and Methods: Twenty-one Class II patients (10 boys; mean age, 11.7 years) who required Herbst therapy with edgewise treatment underwent cone-beam computed tomography (CBCT) before and after treatment. Nineteen Class I control patients (8 boys; mean age, 11.9 years) received edgewise treatment alone. The pressure and velocity of the PA were compared between the groups using CFD based on three-dimensional CBCT images of the PA. Results: The change in oropharyngeal airway velocity in the Herbst group (1.95 m/s) was significantly larger than that in the control group (0.67 m/s). Similarly, the decrease in laryngopharyngeal airway velocity in the Herbst group (1.37 m/s) was significantly larger than that in the control group (0.57 m/s). Conclusion: The Herbst appliance improves ventilation of the oropharyngeal and laryngopharyngeal airways. These results may provide a useful assessment of obstructive sleep apnea treatment during growth

Comparison of dynamic occlusal contacts during chewing between working and balancing sides

Objectives: Mastication is a crucial function for the elderly, and promotes oral health status, cognitive function and the physical constitution. Most reports about occlusion patterns and occlusal glide of adults have reported the jaw movement at the lower incisal point due to easiness of evaluating masticatory performance. The purpose of this study was to test the hypothesis that dynamic occlusal contact area (OCA) during chewing differ for each tooth on the working vs. the balancing chewing side. Design: In thirteen healthy Japanese females, OCA was estimated with a measurement system combining 3-D tracking of mandibular movements with 3-D digitization of tooth shape. Results: The starting of occlusal contact between teeth at working side and balancing side did not differ significantly. In contrast, ending of occlusal contact of teeth at balancing side were markedly longer than that of teeth at working side at lateral incisor, canine, and first premolar. The dynamic sum of OCAs for all teeth was symmetrical around maximum closed position (MCP) when chewing on the working side. In contrast, the dynamic sum of OCA peaked after MCP when chewing on the balancing side. In working and balancing side, sums of maximum OCA at all posterior teeth accounted for 93%, 86% of sum OCA for all teeth at working and balancing sides, respectively. Conclusion: Our result suggested that the hypothesis that dynamic OCA during chewing differ for each tooth on the working vs. the balancing chewing side was not accepted at molars

トクシマケン イシカイ トウニョウビョウ タイサクハン ダイ1ジ ダイ2ジ カツドウ ノ セイカ

Objective : The effectiveness of diabetes prevention programs for the general population in Tokushima Prefecture was investigated. The programs were designed by Tokushima Medical Association’ s（TMA’s）Steering Committee for Diabetes Prevention. Research design and methods : The committee promoted diabetes prevention by disseminating educational messages on diabetes to the general public and medical care providers, and establishing a referral system among public health centers and medical institutes throughout Tokushima Prefecture during the period from ２００４ to ２００９. The outcome of these activities were evaluated by analyzing data from the Prefectural Health and Nutrition Survey in Tokushima conducted in１９９７（n＝ ９９８）,２００３ （n＝１００８） and ２０１０ （n＝１１３０）, and then comparing these results with those of the national survey at the corresponding times. Results : The percentage of subjects with glucose intolerance at the time of initiation of the prevention program in Tokushima tended to increase from １９９７ to ２００３, but was slightly decreased in ２０１０, although the differences were not statistically significant. However, the percentage of subjects with glucose intolerance was significantly increased throughout Japan during the same period. Obesity parameters, physical activity evaluated by the number of steps and the average total energy intake changed favorably in parallel with changes in the prevalence of diabetes during the study period in Tokushima. Conclusion : The diabetes prevention programs initiated by the TMA’s committee may be useful in ameliorating the situation of diabetes in Tokushima Prefecture

Mutations in the C1 element of the insulin promoter lead to diabetic phenotypes in homozygous mice

Genome editing technologies such as CRISPR–Cas9 are widely used to establish causal associations between mutations and phenotypes. However, CRISPR–Cas9 is rarely used to analyze promoter regions. The insulin promoter region (approximately 1,000 bp) directs β cell-specific expression of insulin, which in vitro studies show is regulated by ubiquitous, as well as pancreatic, β cell-specific transcription factors. However, we are unaware of any confirmatory in vivo studies. Here, we used CRISPR–Cas9 technology to generate mice with mutations in the promoter regions of the insulin I (Ins1) and II (Ins2) genes. We generated 4 homozygous diabetic mice with 2 distinct mutations in the highly conserved C1 elements in each of the Ins1 and Ins2 promoters (3 deletions and 1 replacement in total). Remarkably, all mice with homozygous or heterozygous mutations in other loci were not diabetic. Thus, the C1 element in mice is required for Ins transcription in vivo

Gene Expression in Pancreatic Cancer-Like Cells and Induced Pancreatic Stem Cells Generated by Transient Overexpression of Reprogramming Factors

We previously reported that transient overexpression of reprogramming factors can be used to generate induced pluripotent stem (iPS) cells, induced tissue-specific stem (iTS) cells, and fibroblast-like (iF) cells from pancreatic tissue. iF cells have tumorigenic ability and behave similarly to pancreatic cancer cells. In this study, we analyzed gene expression in iF cells and iTS-P cells (iTS cells from pancreatic tissue) via microarray analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of the Mybl2 and Lyn genes, which are reported to be oncogenes, were significantly higher in iF cells than in iTS-P cells. The expression level of Nestin, which is expressed in not only pancreatic progenitor cells but also pancreatic ductal adenocarcinomas, was also higher in iF cells than in iTS-P cells. Itgb6 and Fgf13, which are involved in the pathogenesis of diseases such as cancer, exhibited higher expression levels in iF cells than in iTS-P cells. Unexpectedly, the expression levels of genes related to epithelial-mesenchymal transition (EMT), except Bmp4, were lower in iF cells than in iTS-P cells. These data suggest that the Mybl2, Lyn, Nestin, Itgb6, and Fgf13 genes could be important biomarkers to distinguish iTS-P cells from iF cells