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

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

Novel features of ARS selection in budding yeast Lachancea kluyveri

<p>Abstract</p> <p>Background</p> <p>The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.</p> <p>Results</p> <p>In this study we have isolated and characterized autonomously replicating sequences (ARSs) in <it>Lachancea kluyveri </it>- a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that <it>L. kluyveri </it>ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in <it>Saccharomyces cerevisiae</it>. Moreover, compared with <it>S. cerevisiae </it>and <it>K. lactis</it>, the replication licensing machinery in <it>L. kluyveri </it>seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all <it>S. cerevisiae </it>ARSs tested and most <it>Kluyveromyces lactis </it>ARSs. In contrast, only about half of the <it>L. kluyveri </it>ARSs function in <it>S. cerevisiae </it>and less than 10% function in <it>K. lactis</it>.</p> <p>Conclusions</p> <p>Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.</p

In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications

Recent Advances in <em>In Vivo</em> Genome Editing Targeting Mammalian Preimplantation Embryos

CRISPR-based genome engineering has been widely used for producing gene-modified animals such as mice and rats, to explore the function of a gene of interest and to create disease models. However, it always requires the ex vivo handling of preimplantation embryos, as exemplified by the microinjection of genome editing components into zygotes or in vitro electroporation of zygotes in the presence of genome editing components, and subsequent cultivation of the treated embryos prior to egg transfer to the recipient females. To avoid this ex vivo process, we have developed a novel method called genome-editing via oviductal nucleic acids delivery (GONAD) or improved GONAD (i-GONAD), which enables in situ genome editing of zygotes present in the oviductal lumen of a pregnant female. This technology does not require any ex vivo handling of preimplantation embryos or preparation of recipient females and vasectomized males, all of which are often laborious and time-consuming. In this chapter, recent advances in the development of GONAD/i-GONAD will be described

Induced Tissue-Specific Stem Cells (iTSCs): Their Generation and Possible Use in Regenerative Medicine

Induced tissue-specific stem cells (iTSCs) are partially reprogrammed cells which have an intermediate state, such as progenitors or stem cells. They originate from the de-differentiation of differentiated somatic cells into pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), or from the differentiation of undifferentiated cells. They show a limited capacity to differentiate and a morphology similar to that of somatic cell stem cells present in tissues, but distinct from that of iPSCs and ESCs. iTSCs can be generally obtained 7 to 10 days after reprogramming of somatic cells with Yamanaka’s factors, and their fibroblast-like morphology remains unaltered. iTSCs can also be obtained directly from iPSCs cultured under conditions allowing cellular differentiation. In this case, to effectively induce iTSCs, additional treatment is required, as exemplified by the conversion of iPSCs into naïve iPSCs. iTSCs can proliferate continuously in vitro, but when transplanted into immunocompromised mice, they fail to generate solid tumors (teratomas), implying loss of tumorigenic potential. The low tendency of iTSCs to elicit tumors is beneficial, especially considering applications for regenerative medicine in humans. Several iTSC types have been identified, including iTS-L, iTS-P, and iTS-D, obtained by reprogramming hepatocytes, pancreatic cells, and deciduous tooth-derived dental pulp cells, respectively. This review provides a brief overview of iPSCs and discusses recent advances in the establishment of iTSCs and their possible applications in regenerative medicine