Novel electroporation microchip with field constriction enhances transfection efficiency and survival rates of feline embryos.

This study introduces a low-voltage electroporation microchip designed for transfection in cat embryos, featuring real-time impedance monitoring. The microchip uses a field constriction strategy, which localises the electric field to the membrane region in contact with the micro-orifice, enhancing electroporation efficiency while minimising damage. Embryos were positioned on the orifice, and a series of voltage pulses (10, 15, and 20 V) were applied.

Generation of induced pluripotent stem cells from the Asian bats.

Preservation of native Korean bats is crucial for maintaining ecological balance, as they play a vital role in insect control, pollination, and seed dispersal within their ecosystems. The present study details the establishment of bat induced pluripotent stem cells (BatiPSCs) from two Asian and Korean bats ( and ) using the Sendai Reprogramming Kit. Colonies of BatiPSCs, exhibiting distinctive features, were manually selected and expanded following successful transfection.

Identification -like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9.

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The locus is a well-known safe harbor for human and mouse studies. In this study, we found an -like sequence (p) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the p.

ROCK Inhibitor (Y-27632) Abolishes the Negative Impacts of miR-155 in the Endometrium-Derived Extracellular Vesicles and Supports Embryo Attachment.

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics.

Prime editor-mediated correction of a pathogenic mutation in purebred dogs.

Canine hip dysplasia (HD) is a multifactorial disease caused by interactions between genetic and environmental factors. HD, which mainly occurs in medium- to large-sized dogs, is a disease that causes severe pain and requires surgical intervention. However, the procedure is not straight-forward, and the only way to ameliorate the situation is to exclude individual dogs with HD from breeding programs.

Generation of genome-edited dogs by somatic cell nuclear transfer.

Background: Canine cloning technology based on somatic cell nuclear transfer (SCNT) combined with genome-editing tools such as CRISPR-Cas9 can be used to correct pathogenic mutations in purebred dogs or to generate animal models of disease.

Results: We constructed a CRISPR-Cas9 vector targeting canine DJ-1. Genome-edited canine fibroblasts were established using vector transfection and antibiotic selection.

PRRX1 is a master transcription factor of stromal fibroblasts for myofibroblastic lineage progression.

Although stromal fibroblasts play a critical role in cancer progression, their identities remain unclear as they exhibit high heterogeneity and plasticity. Here, a master transcription factor (mTF) constructing core-regulatory circuitry, PRRX1, which determines the fibroblast lineage with a myofibroblastic phenotype, is identified for the fibroblast subgroup. PRRX1 orchestrates the functional drift of fibroblasts into myofibroblastic phenotype via TGF-β signaling by remodeling a super-enhancer landscape.

Genetic Dissection of CRISPR-Cas9 Mediated Inheritance of Independently Targeted Alleles in Tobacco and Loci.

We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco () alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an -delivered CRISPR-Cas9 system targeting () and () genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.

Hemp Genome Editing-Challenges and Opportunities.

Hemp ( L.) is a multipurpose crop with many important uses including medicine, fibre, food and biocomposites. This plant is currently gaining prominence and acceptance for its valuable applications.

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts.

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock.

Loss of zinc-finger protein 212 leads to Purkinje cell death and locomotive abnormalities with phospholipase D3 downregulation.

Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age.

ZNF746/PARIS promotes the occurrence of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer to cause liver cancer related deaths worldwide. Zinc finger protein 746 (ZNF746), initially identified as a Parkin-interacting substrate (PARIS), acts as a transcriptional repressor of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in Parkinson's disease. As recent studies reported that PARIS is associated with cancer onset, we investigated whether PARIS is associated with HCC.

Author Correction: CRISPR/Cas9-mediated knockout of Mct8 reveals a functional involvement of Mct8 in testis and sperm development in a rat.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CRISRP/Cas9-mediated knockout of Mct8 reveals a functional involvement of Mct8 in testis and sperm development in a rat.

Thyroid hormone (TH) has long been believed to play a minor role in male reproduction. However, evidences from experimental model of thyrotoxicosis or hypothyroidism suggests its role in spermatogenesis. Cellular action of TH requires membrane transport via specific transporters such as monocarboxylate transporter 8 (MCT8).

Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System.

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days.

CRISPR-Cas9-mediated genome editing in apple and grapevine.

The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components.

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins.

The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties.

Nuclear-mitochondrial incompatibility in interorder rhesus monkey-cow embryos derived from somatic cell nuclear transfer.

Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development.

NME1L Negatively Regulates IGF1-Dependent Proliferation of Breast Cancer Cells.

Non-metastatic cells 1 (NME1) or nm23 is the first metastasis suppressor gene discovered. It functions through various enzymatic activities and interacts with many intracellular proteins. The NME1 gene encodes two splicing variants, NME1 and NME1L.

Dimer of arfaptin 2 regulates NF-κB signaling by interacting with IKKβ/NEMO and inhibiting IKKβ kinase activity.

IκB kinases (IKKs) are a therapeutic target due to their crucial roles in various biological processes, including the immune response, the stress response, and tumor development. IKKs integrate various upstream signals that activate NF-κB by phosphorylating IκB and also regulate many proteins related to cell growth and metabolism. Although they function as a heteromeric complex comprised of kinase subunits and an adaptor, these kinases produce distinct cellular responses by phosphorylating different target molecules, suggesting that they may also be regulated in a subtype-specific manner.

Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells.

Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels.

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes.

Oxamflatin improves developmental competence of porcine somatic cell nuclear transfer embryos.

Abstract Aberrant epigenetic nuclear reprogramming of somatic nuclei is a major cause of low success in cloning. It has been demonstrated that treatment of histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos by alteration of epigenetic status. The aim of the present study was to investigate the effect of oxamflatin, a novel HDACi, on the developmental competence of porcine SCNT embryos.

Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre-loxP.

It is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development.