Expression of a single-chain monellin (MNEI) mutant with enhanced stability in transgenic mice milk.

Monellin is a sweet protein that may be used as a safe and healthy sweetener. However, due to its low stability, the application of monellin is currently very limited. Here, we describe a wild-type, a double-sites mutant (E2N/E23A) and a triple-sites mutant (N14A/E23Q/S76Y) of single-chain monellin (MNEI) expressed in transgenic mice milk. Based on enzyme-linked immunoassay (ELISA), Western blot, and sweetness intensity testing, their sweetness and stability were compared. After boiling for 2 min at different pH conditions (2.5, 5.1, 6.8, and 8.2), N14A/E23Q/S76Y-MNEI showed significantly higher sweetness and stability than the wild-type and E2N/E23A-MNEI. These results suggest that N14A/E23Q/S76Y-MNEI shows remarkable potential as a sweetener in the future.

Rare Variant in Metallothionein 1E Increases the Risk of Type 2 Diabetes in a Chinese Population.

OBJECTIVE: To uncover novel targets for the treatment of type 2 diabetes (T2D) by investigating rare variants with large effects in monogenic forms of the disease. RESEARCH DESIGN AND METHODS: We performed whole-exome sequencing in a family with diabetes. We validated the identified gene using Sanger sequencing in additional families and diabetes- and community-based cohorts. Wild-type and variant gene transgenic mouse models were used to study the gene function. RESULTS: Our analysis revealed a rare variant of the metallothionein 1E (MT1E) gene, p.C36Y, in a three-generation family with diabetes. This risk allele was associated with T2D or prediabetes in a community-based cohort. MT1E p.C36 carriers had higher HbA1c levels and greater BMI than those carrying the wild-type allele. Mice with forced expression of MT1E p.C36Y demonstrated increased weight gain, elevated postchallenge serum glucose and liver enzyme levels, and hepatic steatosis, similar to the phenotypes observed in human carriers of MT1E p.C36Y. In contrast, mice with forced expression of MT1E p.C36C displayed reduced weight and lower serum glucose and serum triglyceride levels. Forced expression of wild-type and variant MT1E demonstrated differential expression of genes related to lipid metabolism. CONCLUSIONS: Our results suggest that MT1E could be a promising target for drug development, because forced expression of MT1E p.C36C stabilized glucose metabolism and reduced body weight, whereas MT1E p.C36Y expression had the opposite effect. These findings highlight the importance of considering the impact of rare variants in the development of new T2D treatments.

Aberrant glial activation and synaptic defects in CaMKIIα-iCre and nestin-Cre transgenic mouse models.

Current scientific research is driven by the ability to manipulate gene expression by utilizing the Cre/loxP system in transgenic mouse models. However, artifacts in Cre-driver mouse lines that introduce undesired effects and confound results are increasingly being reported. Here, we show aberrant neuroinflammation and synaptic changes in two widely used Cre-driver mouse models. Neuroinflammation in CaMKIIα-iCre mice was characterized by the activation and proliferation of microglia and astrocytes in synaptic layers of the hippocampus. Increased GFAP and Iba1 levels were observed in hippocampal brain regions of 4-, 8- and 22-month-old CaMKIIα-iCre mice compared to WT littermates. Synaptic changes in NMDAR, AMPAR, PSD95 and phosphorylated CaMKIIα became apparent in 8-month-old CaMKIIα-iCre mice but were not observed in 4-month-old CaMKIIα-iCre mice. Synaptophysin and synaptoporin were unchanged in CaMKIIα-iCre compared to WT mice, suggesting that synaptic alterations may occur in excitatory postsynaptic regions in which iCre is predominantly expressed. Finally, hippocampal volume was reduced in 22-month-old CaMKIIα-iCre mice compared to WT mice. We tested the brains of mice of additional common Cre-driver mouse models for neuroinflammation; the nestin-Cre mouse model showed synaptic changes and astrocytosis marked by increased GFAP+ astrocytes in cortical and hippocampal regions, while the original CaMKIIα-Cre T29-1 strain was comparable to WT mice. The mechanisms underlying abnormal neuroinflammation in nestin-Cre and CaMKIIα-iCre are unknown but may be associated with high levels of Cre expression. Our findings are critical to the scientific community and demonstrate that the correct Cre-driver controls must be included in all studies using these mice.

Common human genetic variants of APOE impact murine COVID-19 mortality.

Clinical outcomes of severe acute respiratory syndrome 2 (SARS-CoV-2) infection are highly heterogeneous, ranging from asymptomatic infection to lethal coronavirus disease 2019 (COVID-19). The factors underlying this heterogeneity remain insufficiently understood. Genetic association studies have suggested that genetic variants contribute to the heterogeneity of COVID-19 outcomes, but the underlying potential causal mechanisms are insufficiently understood. Here we show that common variants of the apolipoprotein E (APOE) gene, homozygous in approximately 3% of the world's population1 and associated with Alzheimer's disease, atherosclerosis and anti-tumour immunity2-5, affect COVID-19 outcome in a mouse model that recapitulates increased susceptibility conferred by male sex and advanced age. Mice bearing the APOE2 or APOE4 variant exhibited rapid disease progression and poor survival outcomes relative to mice bearing the most prevalent APOE3 allele. APOE2 and APOE4 mice exhibited increased viral loads as well as suppressed adaptive immune responses early after infection. In vitro assays demonstrated increased infection in the presence of APOE2 and APOE4 relative to APOE3, indicating that differential outcomes are mediated by differential effects of APOE variants on both viral infection and antiviral immunity. Consistent with these in vivo findings in mice, our results also show that APOE genotype is associated with survival in patients infected with SARS-CoV-2 in the UK Biobank (candidate variant analysis, P = 2.6 × 10-7). Our findings suggest APOE genotype to partially explain the heterogeneity of COVID-19 outcomes and warrant prospective studies to assess APOE genotyping as a means of identifying patients at high risk for adverse outcomes.

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles.

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Applications of and considerations for using CRISPR-Cas9-mediated gene conversion systems in rodents.

Genetic elements that are inherited at super-Mendelian frequencies could be used in a 'gene drive' to spread an allele to high prevalence in a population with the goal of eliminating invasive species or disease vectors. We recently demonstrated that the gene conversion mechanism underlying a CRISPR-Cas9-mediated gene drive is feasible in mice. Although substantial technical hurdles remain, overcoming these could lead to strategies that might decrease the spread of rodent-borne Lyme disease or eliminate invasive populations of mice and rats that devastate island ecology. Perhaps more immediately achievable at moderate gene conversion efficiency, applications in a laboratory setting could produce complex genotypes that reduce the time and cost in both dollars and animal lives compared with Mendelian inheritance strategies. Here, we discuss what we have learned from early efforts to achieve CRISPR-Cas9-mediated gene conversion, potential for broader applications in the laboratory, current limitations, and plans for optimizing this potentially powerful technology.

Metabolic Effects of CCN5/WISP2 Gene Deficiency and Transgenic Overexpression in Mice.

CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 might promote the proliferation and survival of pancreatic ß-cells and thus improve the metabolic profile of the animals. More recently, the roles of endogenous expression of CCN5 and its ectopic, transgenic overexpression on metabolic regulation have been revealed through two reports. Here, we attempt to compare the experimental findings from those studies, side-by-side, in order to further establish its roles in metabolic regulation. Prominent among the discoveries was that a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance, which were further exacerbated upon high-fat diet feeding. On the other hand, the adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. Not all results from these models are consistent; moreover, several important aspects of CCN5 physiology are yet to be explored.

BiP Heterozigosity Aggravates Pathological Deterioration in Experimental Amyotrophic Lateral Sclerosis.

In the present study, we investigated the involvement of the chaperone protein BiP (also known as GRP78 or Hspa5), a master regulator of intracellular proteostasis, in two mouse models of neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). To this end, we used mice bearing partial genetic deletion of the BiP gene (BiP+/- mice), which, for the ALS model, were crossed with mutant SOD1 (mSOD1) transgenic mice to generate mSOD1/BiP+/- double mutant mice. Our data revealed a more intense neurological decline in the double mutants, reflected in a greater deterioration of the neurological score and rotarod performance, with also a reduced animal survival, compared to mSOD1 transgenic mice. Such worsening was associated with higher microglial (labelled with Iba-1 immunostaining) and, to a lesser extent, astroglial (labelled with GFAP immunostaining) immunoreactivities found in the double mutants, but not with a higher loss of spinal motor neurons (labelled with Nissl staining) in the spinal cord. The morphological analysis of Iba-1 and GFAP-positive cells revealed a higher presence of activated cells, characterized by elevated cell body size and shorter processes, in double mutants compared to mSOD1 mice with normal BiP expression. In the case of the PD model, BiP+/- mice were unilaterally lesioned with the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). In this case, however, we did not detect a greater susceptibility to damage in mutant mice, as the motor defects caused by 6-OHDA in the pole test and the cylinder rearing test, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity (labelled with CD68 and GFAP immunostaining) detected in the substantia nigra were of similar magnitude in BiP+/- mice compared with wildtype animals. Therefore, our findings support the view that a dysregulation of the protein BiP may contribute to ALS pathogenesis. As BiP has been recently related to cannabinoid type-1 (CB1) receptor function, our work also opens the door to future studies on a possible link between BiP and the neuroprotective effects of cannabinoids that have been widely reported in this neuropathological context. In support of this possibility, preliminary data indicate that CB1 receptor levels are significantly reduced in mSOD1 mice having partial deletion of BiP gene.

A bioluminescence reporter mouse strain for in vivo imaging of CD8+ T cell localization and function.

CD8+ T cells play a critical role during adaptive immune response, which often change locations and expand or contract in numbers under different states. In the past, many attempts to develop CD8+T cells that express luciferase in vivo have involved the use of viral transduction, which has drawbacks of hardly tracked via detection of luciferase signal in untouched natural states. Here, we generate a transgenic mouse model via CRISPR-mediated genome editing, C57BL/6-CD8aem(IRES-AkaLuci-2A-EGFP) knock-in mice(CD8a-Aka mice), as a novel tool for non-invasive imaging of CD8+ T cells, which expressed a highly sensitive luciferase-Akaluciferase. Our study offers a convenient and robust tool for understanding fundamental CD8+ T cell biology in experimental applications and preclinical translational studies.

Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system.

Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies. We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7). The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Importantly, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes at different developmental stages that may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration. In conclusion, the FUCCI mouse provides a valuable system to study cardiomyocyte cell cycle activity at single cell resolution that can help to decipher the switch from cardiomyocyte proliferation to endoreplication, and to revert this process to facilitate endogenous repair.

Live-cell imaging of microglial interactions with radial glia in transgenic embryonic mouse brains using slice culture.

Microglial dynamics and interactions with nearby radial glia can be visualized in real time in embryonic mouse brain tissue using time-lapse imaging in slice culture. This live-cell imaging protocol can be used to study the morphology and activities of a number of cell types across a variety of brain regions and developmental time points. The advantage of this brain slice culture model is that it allows for the visualization of cellular interactions and movements in real time, especially across embryogenesis. For complete details on the use and execution of this protocol, please refer to Rosin et al. (2021).

Transgenic mouse models of breast cancer.

Transgenic breast cancer mouse models are critical tools for preclinical studies of human breast cancer. Genetic editing of the murine mammary gland allows for modeling of abnormal genetic events frequently found in human breast cancers. Genetically engineered mouse models (GEMMs) of breast cancer employ tissue-specific genetic manipulation for tumorigenic induction within the mammary tissue. Under the transcriptional control of mammary-specific promoters, transgenic mouse models can simulate spontaneous mammary tumorigenesis by expressing one or more putative oncogenes, such as MYC, HRAS, and PIK3CA. Alternatively, the Cre-Lox system allows for tissue-specific deletion of tumor suppressors, such as p53, Rb1, and Brca1, or specific knock-in of putative oncogenes. Thus, GEMMs can be designed to implement one or more genetic events to induce mammary tumorigenesis. Features of GEMMs, such as age of transgene expression, breeding quality, tumor latency, histopathological characteristics, and propensity for local and distant metastasis, are variable and strain-dependent. This review aims to summarize currently available transgenic breast cancer mouse models that undergo spontaneous mammary tumorigenesis upon genetic manipulation, their varying characteristics, and their individual genetic manipulations that model aberrant signaling events observed in human breast cancers.

The diversity of the plasmablast signature across species and experimental conditions: A meta-analysis.

Antibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states.

Orchestrating aquaporin-4 and connexin-43 expression in brain: Differential roles of α1- and ß1-syntrophin.

Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether ß1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of ß1-syntrophin deletion is rather limited, double knockout of α1- and ß1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.

An APP ectodomain mutation outside of the Aß domain promotes Aß production in vitro and deposition in vivo.

Familial Alzheimer's disease (FAD)-linked mutations in the APP gene occur either within the Aß-coding region or immediately proximal and are located in exons 16 and 17, which encode Aß peptides. We have identified an extremely rare, partially penetrant, single nucleotide variant (SNV), rs145081708, in APP that corresponds to a Ser198Pro substitution in exon 5. We now report that in stably transfected cells, expression of APP harboring the S198P mutation (APPS198P) leads to elevated production of Aß peptides by an unconventional mechanism in which the folding and exit of APPS198P from the endoplasmic reticulum is accelerated. More importantly, coexpression of APP S198P and the FAD-linked PS1ΔE9 variant in the brains of male and female transgenic mice leads to elevated steady-state Aß peptide levels and acceleration of Aß deposition compared with age- and gender-matched mice expressing APP and PS1ΔE9. This is the first AD-linked mutation in APP present outside of exons 16 and 17 that enhances Aß production and deposition.

Protocol for behavioral tests using chemogenetically manipulated mice.

Behavioral analyses using mice chemogenetically manipulated by designer receptors exclusively activated by designer drugs (DREADDs) are powerful tools to elucidate neural functions. Here, we describe the detailed protocols for stereotaxic surgery, adeno-associated virus (AAV)-mediated introduction to Gq-DREADDs in mice, and for behavioral testing and analyses related to anxiety, risk assessment, and burying behaviors. A series of these tests are useful in evaluating animal anxiety and their defensive response patterns to potential threats. For complete details on the use and execution of this protocol, please refer to Horii-Hayashi et al. (2021).

Ezh1 regulates expression of Cpg15/Neuritin in mouse cortical neurons.

Immature neurons undergo morphological and physiological maturation in order to establish neuronal networks. During neuronal maturation, a large number of genes change their transcriptional levels, and these changes may be mediated by chromatin modifiers. In this study, we found that the level of Ezh1, a component of Polycomb repressive complex 2 (PRC2), increases during neuronal maturation in mouse neocortical culture. In addition, conditional knockout of Ezh1 in post-mitotic excitatory neurons leads to downregulation of a set of genes related to neuronal maturation. Moreover, the locus encoding Cpg15/Neuritin (Nrn1), which is regulated by neuronal activity and implicated in stabilization and maturation of excitatory synapses, is a direct target of Ezh1 in cortical neurons. Together, these results suggest that elevated expression of Ezh1 contributes to maturation of cortical neurons.

Transgenic inhibition of interleukin-6 trans-signaling does not prevent skeletal pathologies in mucolipidosis type II mice.

Severe skeletal alterations are common symptoms in patients with mucolipidosis type II (MLII), a rare lysosomal storage disorder of childhood. We have previously reported that progressive bone loss in a mouse model for MLII is caused by an increased number of bone-resorbing osteoclasts, which is accompanied by elevated expression of the cytokine interleukin-6 (IL-6) in the bone microenvironment. In the present study we addressed the question, if pharmacological blockade of IL-6 can prevent the low bone mass phenotype of MLII mice. Since the cellular IL-6 response can be mediated by either the membrane-bound (classic signaling) or the soluble IL-6 receptor (trans-signaling), we first performed cell culture assays and found that both pathways can increase osteoclastogenesis. We then crossed MLII mice with transgenic mice expressing the recombinant soluble fusion protein sgp130Fc, which represents a natural inhibitor of IL-6 trans-signaling. By undecalcified histology and bone-specific histomorphometry we found that high circulating sgp130Fc levels do not affect skeletal growth or remodeling in wild-type mice. Most importantly, blockade of IL-6 trans-signaling did neither reduce osteoclastogenesis, nor increase bone mass in MLII mice. Therefore, our data clearly demonstrate that the bone phenotype of MLII mice cannot be corrected by blocking the IL-6 trans-signaling.

Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice.

Taking advantage of their ability to integrate their genomes into the host genome, lentiviruses have been used to rapidly produce transgenic mice in biomedical research. In most cases, transgenes delivered by lentiviral vectors have resisted silencing mediated by epigenetic modifications in mice. However, some studies revealed that methylation caused decreased transgene expression in mice. Therefore, there is conflicting evidence regarding the methylation-induced silencing of transgenes delivered by lentiviral transduction in mice. In this study, we present evidence that the human TTR transgene was silenced by DNA methylation in the liver of a transgenic mouse model generated by lentiviral transduction. The density of methylation on the transgene was increased during reproduction, and the expression of the transgene was completely silenced in mice of the F2 generation. Interestingly, 5-azacytidine (5-AzaC), a methyltransferase inhibitor, potently reactivated the silenced genes in neonatal mice whose hepatocytes were actively proliferating and led to stable transgene expression during development. However, 5-AzaC did not rescue liver transgene expression when administered to adult mice. Moreover, 5-AzaC at the given dose had low developmental toxicity in the newborn mice. In summary, we demonstrate the methylation-induced silencing of an exogenous gene in the liver of a mouse model generated by lentiviral transduction and show that the silenced transgene can be safely and efficiently reactivated by 5-AzaC treatment, providing an alternative way to obtain progeny with stable transgene expression in the case of the methylation of exogenous genes in transgenic mice generated by lentiviral transduction.