Efficient gene editing of pig embryos by combining electroporation and lipofection.

Background And Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences.

Materials And Methods: Six gRNAs (gRNA1-6) with different target sequences were designed to target APN.

[Invasive Ductal Carcinoma with Total Infarction and Necrosis-Case Report and Literature Review].

A 71-year-old woman visited our hospital with pain and itching in her left breast which had commenced the day before admission. On palpation, we detected a 2.0 cm nodule, indicative of an elastic and hard tumor located centrally in the left breast.

Impact of redox status of donor cows before superovulation treatment on in vivo embryo production.

This study investigated the relationship between oxidation and antioxidation parameters before superovulation (SOV) treatment and embryo recovery in donor cows. The relative redox status of the 61 donor cows was evaluated based on the median values of diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) measurements (d-ROMs of 100 U.CARR; BAP of 2413  mol L ) before SOV treatment.

Retinal pigment epithelium-specific ablation of GPx4 in adult mice recapitulates key features of geographic atrophy in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly population, particularly the late-stage of dry AMD known as geographic atrophy (GA), lacks effective treatment options. Genetic mouse models of AMD have revealed the significance of impaired lipid metabolism and anti-oxidative capacity in early/intermediate stage of AMD, but remains unclear in GA that severely damages visual function. Here, to investigate the potential relevance of peroxidized lipids in RPE for late-stage dry AMD, GPx4 mice underwent subretinal injections of RPE-specific AAV-Cre vector or control AAV vector.

Effects of ergothioneine supplementation on meiotic competence and porcine oocyte development.

Background And Aim: The antioxidant effects of ergothioneine (EGT) on culture of porcine zygote are not well established. The study investigated the impact of EGT supplementation on meiotic competence and development of porcine oocytes.

Materials And Methods: The impact of EGT concentrations (0, 5, 10, 25, 50, and 100 μM) during maturation (IVM) on the progression of meiotic maturation, fertilization, and blastocyst formation in porcine oocytes was assessed.

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h.

Visual prognostic factors following surgery for stage 3 idiopathic epiretinal membrane by Govetto classification.

Purpose: To investigate the visual prognosis of metamorphopsia in patients undergoing surgery for stage 3 idiopathic epiretinal membrane (ERM) by Govetto classification using preoperative optical coherence tomography (OCT) parameters.

Study Design: Retrospective clinical study.

Method: This study included 45 eyes of 45 patients with a minimum follow-up period of 3 months.

Quality and fertilizing ability of frozen-thawed porcine sperm separated using a migration sedimentation method.

We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device.

Effects of centrifugation treatment before electroporation on gene editing in pig embryos.

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation.

Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates.

A Recql5 mutant facilitates complex CRISPR/Cas9-mediated chromosomal engineering in mouse zygotes.

Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions.

Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency.

Development of porcine embryos cultured in media irradiated with ultraviolet-C.

Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively.

Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen.

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.

Selection of spermatozoa with high motility and quality from bovine frozen-thawed semen using the centrifuge-free device.

This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method.

RECENT TRENDS IN THE CUMULATIVE INCIDENCE AND INTERVENTION PATTERNS OF RETINOPATHY OF PREMATURITY IN JAPAN: A Multicenter Analysis, 2011-2020.

Purpose: To investigate recent trends in the cumulative incidence and treatment patterns of retinopathy of prematurity (ROP) in Japan.

Methods: A retrospective multicenter cohort was conducted from 2011 to 2020 using the Diagnosis Procedure Combination inpatient database. Preterm newborns with birth weight <2,500 g were categorized by birth weight.

Vitrified before and after genome editing via electroporation.

Background: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation.

Objective: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes.

Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos.

The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization.

Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system.

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin.

Mosaic Mutation on Tumour Development in Pigs: A Case Study.

Pigs rarely develop cancer; however, tumour protein p53 ()-modified pigs may have an increased incidence of cancer. In this study, two pigs with mosaic mutations induced by gene editing were compared to determine the role of the wild-type sequence in tumorigenesis and to speculate how amino acid changes in TP53 sequences are related to tumorigenesis. The pig without tumours had a wild-type sequence and a 1-bp deletion in the sequence that resulted in a premature stop codon.

Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene.

Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis.

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.

Background: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models.

Effects of curcumin supplementation on quality of porcine spermatozoa irradiated with ultraviolet-C at 228 nm during liquid preservation.

It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces.