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The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10 days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6 months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock. [ABSTRACT FROM AUTHOR]

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46 h with an interval of 2 h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised β-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β‐galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH‐SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock‐out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP‐CHOP websites, and high‐efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH‐SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X‐gal and Periodic acid‐Schiff (PAS) staining, and qPCR were used to confirm the knock‐out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock‐out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH‐SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non‐transfected cell, X‐gal staining resulted in no blue color in GLB1 gene knock‐out cells indicating the absence of β‐galactosidase enzyme activity in these cells. Real‐time PCR (qPCR) results confirmed the RNA‐Seq analysis outcomes on the GEO data set and following the GLB1 gene knock‐out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1‐NEU1‐CTSA complex gene was involved in suppressed proliferation and invasion ability of knock‐out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH‐SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease. Significance statement: This study concerns the modeling of GM1 gangliosidosis by knocking‐out the GLB1 gene in human nerve cells and using CRISPR/Cas9 technology. [ABSTRACT FROM AUTHOR]

Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo- derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and β4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo -derived embryos showing efficient multiple gene editing for GGTA1 and GHR. • A comparison of three methodologies based on the introduction of CRISPR-Cas9 complex before (IVF), during (ICSI-MGE), and after fertilization (in vivo) for the production of genetically edited pigs was performed. • ICSI mediated gene editing proved to be an efficient technique for obtaining homozygous biallelic mutated pig embryos, similar to the microinjection of oocytes followed by IVF. • Live births were obtained from in vivo -derived embryos showing efficient multiple gene editing for GGTA1 and GHR genes. [ABSTRACT FROM AUTHOR]

Gene knock‐out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET‐mediated demethylation, more long‐lived mouse models continue to be developed to further define its organismic role. The Polb‐KO mouse was the first of the Cre‐mediated tissue‐specific KO mouse models. This technology was exploited to investigate roles for Polβ in V(D)J recombination (variable‐diversity‐joining rearrangement), DNA demethylation, gene complementation, SPO11‐induced DNA double‐strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin‐induced DNA damage, and cancer onset. The revolution in knock‐in (KI) mouse models was made possible by CRISPR/cas9‐mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polβ. Such KI mouse models have helped uncover the importance of key Polβ active site residues or specific Polβ enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polβ protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polβ protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polβ. [ABSTRACT FROM AUTHOR]

The malfunctioning of the brain synucleins is associated with pathogenesis of Parkinson's disease. Synucleins' ability to modulate various pre-synaptic processes suggests their modifying effects on the electroencephalogram (EEG) recorded from different brain structures. Disturbances in interrelations between them are critical for the onset and evolution of neurodegenerative diseases. Recently, we have shown that, in mice lacking several synucleins, differences between the frequency spectra of EEG from different brain structures are correlated with specificity of synucleins' combinations. Given that EEG spectra are indirect characteristics of inter-structural relations, in this study, we analyzed a coherence of instantaneous values for EEGs recorded from different structures as a direct measure of "functional connectivity" between them. Methods: EEG data from seven groups of knock-out (KO) mice with combined deletions of alpha, beta, and gamma synucleins versus a group of wild-type (WT) mice were compared. EEG coherence was estimated between the cortex (MC), putamen (Pt), ventral tegmental area (VTA), and substantia nigra (SN) in all combinations. Results: EEG coherence suppression, predominantly in the beta frequency band, was observed in KO mice versus WT littermates. The suppression was minimal in MC-Pt and VTA-SN interrelations in all KO groups and in all inter-structural relations in mice lacking either all synucleins or only beta synuclein. In other combinations of deleted synucleins, significant EEG coherence suppression in KO mice was dominant in relations with VTA and SN. Conclusion: Deletions of the synucleins produced significant attenuation of intra-cerebral EEG coherence depending on the imbalance of different types of synucleins. [ABSTRACT FROM AUTHOR]

Substance use disorders are heritable disorders characterized by compulsive drug use, the biological mechanisms for which remain largely unknown. Genetic correlations reveal that predisposing drug‐naïve phenotypes, including anxiety, depression, novelty preference and sensation seeking, are predictive of drug‐use phenotypes, thereby implicating shared genetic mechanisms. High‐throughput behavioral screening in knockout (KO) mice allows efficient discovery of the function of genes. We used this strategy in two rounds of candidate prioritization in which we identified 33 drug‐use candidate genes based upon predisposing drug‐naïve phenotypes and ultimately validated the perturbation of 22 genes as causal drivers of substance intake. We selected 19/221 KO strains (8.5%) that had a difference from control on at least one drug‐naïve predictive behavioral phenotype and determined that 15/19 (~80%) affected the consumption or preference for alcohol, methamphetamine or both. No mutant exhibited a difference in nicotine consumption or preference which was possibly confounded with saccharin. In the second round of prioritization, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two‐bottle choice and ethanol drinking‐in‐the‐dark protocols. We identified 15/401 KO strains (3.7%, which included one gene from the first cohort) that differed most from controls for the predisposing phenotypes. 8 of 15 gene deletions (53%) affected intake or preference for alcohol, methamphetamine or both. Using multivariate and bioinformatic analyses, we observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction‐related processes further. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) genome editing technology, derived from the adaptive immune system of bacteria, has now been recognized as one of the most useful genetic tools in genome engineering. Short stretch of RNA guides the Cas enzyme to precise locations within the target genome of living organisms to accurately modify and control the functions of genes with high accuracy. Cas-mediated genome editing is scalable, practically feasible, and permits the research community to expound the functional organization of the hereditary material and helps to understand the relationship between genetic variations and biological phenotypes. Although CRISPR/Cas system has opened many possibilities in the field of genetic engineering, it still has some shortcomings, such as constraints of the protospacer-adjacent motif (PAM) sequence, relatively larger size of the Cas9 gene and higher chances of off-targeting. To overcome these shortcomings, many new Cas9 variants have been engineered with improved features such as specificity and editing efficacy. Anticipated traits can be developed in plants with high utility in agriculture. For the past decade, this editing system has made significant progress and has been widely used in genome editing to create gene knock-ins and knock-outs. Genome editing is mediated either by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanism, consequently resulting in desired mutations that directly enhance plant performance. This paper summarizes the new variants of the CRISPR/Cas system and applications of the CRISPR system in plant science for the enhancement of various attributes such as yield, quality, nutritional factors, stress tolerance, and herbicide tolerance through knock-in and knock-out strategies. [ABSTRACT FROM AUTHOR]

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products. [ABSTRACT FROM AUTHOR]

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised β-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

With the advancement of genomic engineering and genetic modification techniques, the uptake of computational tools to design guide RNA increased drastically. Searching for genomic targets to design guides with maximum on-target activity (efficiency) and minimum off-target activity (specificity) is now an essential part of genome editing experiments. Today, a variety of tools exist that allow the search of genomic targets and let users customize their search parameters to better suit their experiments. Here we present an overview of different ways to visualize these searched CRISPR target sites along with specific downstream information like primer design, restriction enzyme activity and mutational outcome prediction after a double-stranded break. We discuss the importance of a good visualization summary to interpret information along with different ways to represent similar information effectively. [ABSTRACT FROM AUTHOR]

The malfunctioning of the brain synucleins is associated with pathogenesis of Parkinson’s disease. Synucleins’ ability to modulate various pre-synaptic processes suggests their modifying effects on the electroencephalogram (EEG) recorded from different brain structures. Disturbances in interrelations between them are critical for the onset and evolution of neurodegenerative diseases. Recently, we have shown that, in mice lacking several synucleins, differences between the frequency spectra of EEG from different brain structures are correlated with specificity of synucleins’ combinations. Given that EEG spectra are indirect characteristics of inter-structural relations, in this study, we analyzed a coherence of instantaneous values for EEGs recorded from different structures as a direct measure of “functional connectivity” between them. Methods: EEG data from seven groups of knock-out (KO) mice with combined deletions of alpha, beta, and gamma synucleins versus a group of wild-type (WT) mice were compared. EEG coherence was estimated between the cortex (MC), putamen (Pt), ventral tegmental area (VTA), and substantia nigra (SN) in all combinations. Results: EEG coherence suppression, predominantly in the beta frequency band, was observed in KO mice versus WT littermates. The suppression was minimal in MC-Pt and VTA-SN interrelations in all KO groups and in all inter-structural relations in mice lacking either all synucleins or only beta synuclein. In other combinations of deleted synucleins, significant EEG coherence suppression in KO mice was dominant in relations with VTA and SN. Conclusion: Deletions of the synucleins produced significant attenuation of intra-cerebral EEG coherence depending on the imbalance of different types of synucleins.

Problems associated with the abuse of amphetamine-type stimulants, including methamphetamine (MA), pose serious health and socioeconomic issues world-wide. While it is well-established that MA's psychopharmacological effects involve interactions with monoamine neurotransmission, accumulating evidence from animal models implicates dysregulated glutamate in MA addiction vulnerability and use disorder. Recently, we discovered an association between genetic vulnerability to MA-taking and increased expression of the glutamate receptor scaffolding protein Homer2 within both the shell and core subregions of the nucleus accumbens (NAC) and demonstrated a necessary role for Homer2 within the shell subregion in MA reward and reinforcement in mice. This report extends our earlier work by interrogating the functional relevance of Homer2 within the NAC core for the conditioned rewarding and reinforcing properties of MA. C57BL/6J mice with a virus-mediated knockdown of Homer2b expression within the NAC core were first tested for the development and expression of a MA-induced conditioned place-preference/CPP (four pairings of 2 mg/kg MA) and then were trained to self-administer oral MA under operant-conditioning procedures (5-80 mg/L). Homer2b knockdown in the NAC core augmented a MA-CPP and shifted the dose-response function for MA-reinforced responding, above control levels. To determine whether Homer2b within NAC subregions played an active role in regulating MA reward and reinforcement, we characterized the MA phenotype of constitutive Homer2 knockout (KO) mice and then assayed the effects of virus-mediated overexpression of Homer2b within the NAC shell and core of wild-type and KO mice. In line with the results of NAC core knockdown, Homer2 deletion potentiated MA-induced CPP, MA-reinforced responding and intake, as well as both cue- and MA-primed reinstatement of MA-seeking following extinction. However, there was no effect of Homer2b overexpression within the NAC core or the shell on the KO phenotype. These data provide new evidence indicating a globally suppressive role for Homer2 in MA-seeking and MA-taking but argue against specific NAC subregions as the neural loci through which Homer2 actively regulates MA addiction-related behaviors.

Acute kidney injury is associated with increased risk of heart failure and mortality. This study demonstrates that acute kidney injury induces remote cardiac dysfunction, damage, injury, and fibrosis via a galectin-3 (Gal-3) dependent pathway. Gal-3 originates from bone marrow-derived immune cells. Cardiac damage could be prevented by blocking this pathway.

Acute kidney injury is associated with increased risk of heart failure and mortality. This study demonstrates that acute kidney injury induces remote cardiac dysfunction, damage, injury, and fibrosis via a galectin-3 (Gal-3) dependent pathway. Gal-3 originates from bone marrow-derived immune cells. Cardiac damage could be prevented by blocking this pathway.

Genome editing, as exemplified by the CRISPR/Cas9 system, has recently been employed to effectively generate genetically modified animals and cells for the purpose of gene function analysis and disease model creation. There are at least four ways to induce genome editing in individuals: the first is to perform genome editing at the early preimplantation stage, such as fertilized eggs (zygotes), for the creation of whole genetically modified animals; the second is at post-implanted stages, as exemplified by the mid-gestational stages (E9 to E15), for targeting specific cell populations through in utero injection of viral vectors carrying genome-editing components or that of nonviral vectors carrying genome-editing components and subsequent in utero electroporation; the third is at the mid-gestational stages, as exemplified by tail-vein injection of genome-editing components into the pregnant females through which the genome-editing components can be transmitted to fetal cells via a placenta-blood barrier; and the last is at the newborn or adult stage, as exemplified by facial or tail-vein injection of genome-editing components. Here, we focus on the second and third approaches and will review the latest techniques for various methods concerning gene editing in developing fetuses. [ABSTRACT FROM AUTHOR]

A high salt intake causes hemodynamic changes and promotes immune response through cell activation and cytokine production, leading to pro-inflammatory conditions. Transgenic Tff3−/− knock-out mice (TFF3ko) (n = 20) and wild-type mice (WT) (n = 20) were each divided into the (1) low-salt (LS) group and (2) high-salt (HS) group. Ten-week-old animals were fed with standard rodent chow (0.4% NaCl) (LS) or food containing 4% NaCl (HS) for one week (7 days). Inflammatory parameters from the sera were measured by Luminex assay. The integrin expression and rates of T cell subsets of interest from the peripheral blood leukocytes (PBLs) and mesenteric lymph nodes (MLNs) were measured using flow cytometry. There was a significant increase in high-sensitivity C reactive protein (hsCRP) only in the WT mice following the HS diet, while there were no significant changes in the serum levels of IFN-γ, TNF-α, IL-2, IL-4, or IL-6 as a response to treatment in either study groups. The rates of CD4+CD25+ T cells from MLNs decreased, while CD3+γδTCR+ from peripheral blood increased following the HS diet only in TFF3ko. γδTCR expressing T cell rates decreased in WT following the HS diet. The CD49d/VLA-4 expression decreased in the peripheral blood leukocytes in both groups following the HS diet. CD11a/LFA-1 expression significantly increased only in the peripheral blood Ly6C−CD11ahigh monocytes in WT mice following salt loading. In conclusion, salt-loading in knock-out mice caused a lower level of inflammatory response compared with their control WT mice due to gene depletion. [ABSTRACT FROM AUTHOR]

Simple Summary: The application of CRISPR/Cas9 genome editing in fish is limited to date. An easy-to-use, economical and effective genome editing system is greatly needed. In this study, we established an integrated pCas9-U6sgRNA plasmid system, the sgRNA of which was driven by U6 promoters from different fish species. Taking medaka as an example, our results suggest that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) can very effectively mediate gene knock-out in medaka cultured cells, but not by exogenous U6 promoter. The gene editing efficiency of pCas9-mU6sgRNA was up to 93.7% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA can effectively mediate gene knock-out in vivo as well. Furthermore, the gene knock-in at a specific site was efficiently achieved in vitro as well as in vivo through application of pCas9-mU6sgRNA. In conclusion, we have successfully developed a simple, low-cost and effective CRISPR/Cas9 system suitable for medaka gene knock-out and knock-in at a specific site in vitro and in vivo. This study provides an insight into other fish gene manipulation and greatly promotes functional gene studies. Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis. [ABSTRACT FROM AUTHOR]

Abstract Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Three decades following the introduction of the first Rb knockout (KO) mouse model, the role of this critical protein in regulating brain development during embryogenesis and beyond remains a major scientific interest. Rb is a tumor suppressor gene known as the master regulator of the G1/S checkpoint and control of cell cycle progression in stem and progenitor cells, but also their differentiated progeny. Here, we review the recent literature about the various Rb conditional Knockout (cKO) and inducible Knockout (iKO) models studied thus far, highlighting how findings should always be interpreted in light of the model and context under inquiry especially when studying the role of Rb in neuronal survival. There is indeed evidence of age-specific, cell type-specific and region-specific effects following Rb KO in the embryonic and the adult mouse brain. In terms of modeling neurodegenerative processes in human diseases, we discuss cell cycle re-entry (CCE) as a candidate mechanism underlying the increased vulnerability of Rb-deficient neurons to cell death. Notably, mouse models may limit the extent to which CCE due to Rb inactivation can mimic the pathological course of these disorders, such as Alzheimer's disease. These remarks ought to be considered in future research when studying the consequences of Rb inactivation on neuronal generation and survival in rodents and their corresponding clinical significance in humans.

ABSTRACTBCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Genetic manipulation is one of the indispensable techniques to examine gene functions both in vitro and in vivo. In particular, cardiovascular phenotypes such as blood pressure cannot be evaluated in vitro system, necessitating the creation of transgenic or gene-targeted knock-out and knock-in experimental animals to understand the pathophysiological roles of specific genes on the disease conditions. Although genome-wide association studies (GWAS) in various human populations have identified multiple genetic variations associated with increased risk for hypertension and/or its complications, the causal links remain unresolved. Genome-editing technologies can be applied to many different types of cells and organisms for creation of knock-out/knock-in models. In the post-GWAS era, it may be more worthwhile to validate pathophysiological implications of the risk variants and/or candidate genes by creating genome-edited organisms. [ABSTRACT FROM AUTHOR]

Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'. [ABSTRACT FROM AUTHOR]

Abstract Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. Results The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

UnlabelledHow the integrity of laminar structures in the postnatal brain is maintained impacts neuronal functions. Ndel1, the mammalian homolog of NuDE from the filamentous fungus Aspergillus nidulans, is an atypical microtubule (MT)-associated protein that was initially investigated in the contexts of neurogenesis and neuronal migration. Constitutive knock-out mice for Ndel1 are embryonic lethal, thereby necessitating the creation a conditional knock-out to probe the roles of Ndel1 in postnatal brains. Here we report that CA1 pyramidal neurons from mice postnatally lacking Ndel1 (Ndel1 conditional knock-out) exhibit fragmented MTs, dendritic/synaptic pathologies, are intrinsically hyperexcitable and undergo dispersion independently of neuronal migration defect. Secondary to the pyramidal cell changes is the decreased inhibitory drive onto pyramidal cells from interneurons. Levels of the glycoprotein Reelin that regulates MTs, neuronal plasticity, and cell compaction are significantly reduced in hippocampus of mutant mice. Strikingly, a single injection of Reelin into the hippocampus of Ndel1 conditional knock-out mice ameliorates ultrastructural, cellular, morphological, and anatomical CA1 defects. Thus, Ndel1 and Reelin contribute to maintain postnatal CA1 integrity.Significance statementThe significance of this study rests in the elucidation of a role for Nde1l and Reelin in postnatal CA1 integrity using a new conditional knock-out mouse model for the cytoskeletal protein Ndel1, one that circumvents the defects associated with neuronal migration and embryonic lethality. Our study serves as a basis for understanding the mechanisms underlying postnatal hippocampal maintenance and function, and the significance of decreased levels of Ndel1 and Reelin observed in patients with neurological disorders.

Background: Development of neurodegeneration in older people has been associated with microglial cell activation triggered by systemic infection. We hypothesize that α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulation of this process.Methods: 8- to 10-week-old male wild-type (WT) and α7nAChR knock-out (α7nAChR-/-) mice were intraperitoneally inoculated with live Escherichia (E.) coli or saline. After inoculation, all mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h and killed at 2 or 3 days. The microglial response was characterized by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry. To quantify inflammatory response, mRNA expression of pro- and anti-inflammatory mediators was measured in brain and spleen.Results: We observed no differences in Iba-1 positive cell number or morphology and flow cytometry (CD11b, CD45 and CD14) of microglial cells between WT and α7nAChR-/- mice after systemic infection. Infected α7nAChR-/- mice showed significantly higher mRNA expression in brain for tumor necrosis factor alpha (TNF-α) at day 2 and 3, interleukin 6 (IL-6) at day 2 and monocyte chemotactic protein 1 (MCP-1) and suppressor of cytokine signaling 1 (SOCS1) at day 3, there was significantly lower mRNA expression in brain for mitogen-activated protein kinase 1 (MAPK1) at day 2 and 3, high-mobility group 1 (HMGB-1) and CD11b at day 2, and deubiquitinase protein A20 (A20) at day 3 compared to infected WT mice.Interpretation: Loss of function of α7nAChR during systemic infection led to an increased expression of TNF-α and IL-6 in brain after systemic infection with E. coli, but not to distinct differences in microglial cell number or morphological activation of microglia. [ABSTRACT FROM AUTHOR]

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100–200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon–intron structure, with a 70–80% success rate.

Rabbits have been domesticated for meat, wool, and fur production, and have also been cherished as a companion, artistic inspiration, and an experimental model to study many human diseases. In the present study, the muscle mass negative regulator gene myostatin (MSTN) was knocked out in rabbits at two novel sites in exon3, and the function of these mutations was determined in subsequent generations. The prominent double muscle phenotype with hyperplasia or hypertrophy of muscle fiber was observed in the MSTN-KO rabbits, and a similar phenotype was confirmed in the F1 generation. Moreover, the average weight of 80-day-old MSTN-KO rabbits (2,452 ± 63 g) was higher than that of wild-type rabbits (2,393.2 ± 106.88 g), and also the bodyweight of MSTN-KO rabbits (3,708 ± 43.06g) was significantly higher (P < 0.001) at the age of 180 days than wild-type (WT) rabbits (3,224 ± 48.64g). In MSTN-KO rabbits, fourteen rabbit pups from the F1 generation and thirteen from the F2 generation stably inherited the induced MSTN gene mutations. Totally, 194 pups were produced in the F1 generation of which 49 were MSTN-KO rabbits, while 47 pups were produced in the F2 generation of which 20 were edited rabbits, and the ratio of edited to wild-type rabbits in the F2 generation was approximately 1:1. Thus, we successfully generated a heritable double muscle buttocks rabbits via myostatin mutation with CRISPR/Cas9 system, which could be valuable in rabbit's meat production and also a useful animal model to study the development of muscles among livestock species and improve their important economic traits as well as the human muscle development-related diseases.

Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by in vitro differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan in vitro, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Knock-out generation by indel mutation in primary human keratinocytes using nucleofection of ribonucleoprotein (RNP) complex Basic Protocol 2: Deletion of specific genomic region using RNPs via nucleofection Basic Protocol 3: Use of homology-directed repair system to introduce site-specific mutations., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrPC), PrPSc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies. [ABSTRACT FROM AUTHOR]

Background: The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results: We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions: Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC. [ABSTRACT FROM AUTHOR]

Introduction of a gene to mesenchymal stem cells (MSCs) is a well-known strategy to purposely manipulate the cell fate and further enhance therapeutic performance in cell-based therapy. Viral and chemical approaches for gene delivery interfere with differentiation potential. Although microinjection as a physical delivery method is commonly used for transfection, its influence on MSC cell fate is not fully understood. The current study aimed to evaluate the effects of four nonviral gene delivery methods on stem cell multi-potency. The four delivery methods are robotic microinjection, polyethylenimine (PEI), cationic liposome (cLipo), and calcium phosphate nanoparticles (CaP). Among the four methods, microinjection has exhibited the highest transfection efficiency of ∼60%, while the three others showed lower efficiency of 10-25%. Robotic microinjection preserved fibroblast-like cell morphology, stress fibre intactness, and mature focal adhesion complex, while PEI caused severe cytotoxicity. No marked differentiation bias was observed after microinjection and cLipo treatment. By contrast, CaP-treated MSCs exhibited excessive osteogenesis, while PEI-treated MSCs showed excessive adipogenesis. Robotic microinjection system was used to inject the CRISPR/Cas9-encoding plasmid to knock out PPARγ gene in MSCs, and the robotic microinjection did not interfere with PPARγ function in differentiation commitment. Meanwhile, the bias in osteo-adipogenic differentiation exhibited in CaP and PEI-treated MSCs after PPARγ knockout via chemical carriers. Our results indicate that gene delivery vehicles variously disturb MSCs differentiation and interfere with exogenous gene function. Our findings further suggest that robotic microinjection offers a promise of generating genetically modified MSCs without disrupting stem cell multi-potency and therapeutic gene function. [ABSTRACT FROM AUTHOR]

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies. [ABSTRACT FROM AUTHOR]

mRNAs have been found to undergo substantial selective degradation during the late stages of spermiogenesis. However, the mechanisms regulating this biological process are unknown. In this report, we have identified Tex13a, a spermatid-specific gene that interacts with the CCR4–NOT complex and is implicated in the targeted degradation of mRNAs encoding particular structural components of sperm. Deletion of Tex13a led to a delayed decay of these mRNAs, lowered the levels of house-keeping genes, and ultimately lowered several key parameters associated with the control of sperm motility, such as the path velocity (VAP, average path velocity), track speed (VCL, velocity curvilinear), and rapid progression.

We show how to price and replicate a variety of barrier-style claims written on the log price X and quadratic variation 〈 X 〉 of a risky asset. Our framework assumes no arbitrage, frictionless markets and zero interest rates. We model the risky asset as a strictly positive continuous semimartingale with an independent volatility process. The volatility process may exhibit jumps and may be non-Markovian. As hedging instruments, we use only the underlying risky asset, zero-coupon bonds, and European calls and puts with the same maturity as the barrier-style claim. We consider knock-in, knock-out and rebate claims in single and double barrier varieties. [ABSTRACT FROM AUTHOR]

Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene‐editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome‐engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome‐editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome‐editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low‐cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as "improved GONAD (i‐GONAD)" for mice, and "rat improved GONAD (rGONAD)" for rats. [ABSTRACT FROM AUTHOR]

Hyperphosphorylation and accumulation of tau aggregates are prominent features in tauopathies, including Alzheimer's disease, but the impact of loss of tau function on synaptic and cognitive deficits remains poorly understood. We report that old (19-20 months; OKO) but not middle-aged (8-9 months; MKO) tau knock-out mice develop Morris Water Maze (MWM) deficits and loss of hippocampal acetylated α-tubulin and excitatory synaptic proteins. Mild motor deficits and reduction in tyrosine hydroxylase (TH) in the substantia nigra were present by middle age, but did not affect MWM performance, whereas OKO mice showed MWM deficits paralleling hippocampal deficits. Deletion of tau, a microtubule-associated protein (MAP), resulted in increased levels of MAP1A, MAP1B, and MAP2 in MKO, followed by loss of MAP2 and MAP1B in OKO. Hippocampal synaptic deficits in OKO mice were partially corrected with dietary supplementation with docosahexaenoic acid (DHA) and both MWM and synaptic deficits were fully corrected by combining DHA with α-lipoic acid (ALA), which also prevented TH loss. DHA or DHA/ALA restored phosphorylated and total GSK3β and attenuated hyperactivation of the tau C-Jun N-terminal kinases (JNKs) while increasing MAP1B, dephosphorylated (active) MAP2, and acetylated α-tubulin, suggesting improved microtubule stability and maintenance of active compensatory MAPs. Our results implicate the loss of MAP function in age-associated hippocampal deficits and identify a safe dietary intervention, rescuing both MAP function and TH in OKO mice. Therefore, in addition to microtubule-stabilizing therapeutic drugs, preserving or restoring compensatory MAP function may be a useful new prevention strategy.