Adult Mouse Tissues Research Articles

Comorbid epilepsy in autism spectrum disorder: Implications of postnatal inflammation for brain excitability.

In different cohorts, 5%-30% of individuals with autism spectrum disorder (ASD) also have epilepsy. The high co-occurrence of these disorders suggests that a common mechanistic link may exist. The underlying pathophysiology of this comorbidity remains unknown. To investigate the mechanism(s) involved in the pathogenesis of ASD and epilepsy, we developed and validated a novel mouse model that concurrently exhibits hallmark features of both disorders. We utilized inbred BTBR T+ Itpr3tf/J (BTBR) mice that exhibit the core behavioral characteristics of ASD (ie, impaired sociability, altered vocalizations, and restricted interests). BTBR mice received a lipopolysaccharide (LPS) or sterile saline injection at postnatal day (P)7, P14, or P21. Cytokine expression was analyzed for interleukin (IL)-1β, IL-10, IL-6, and tumor necrosis factor α in brain tissue of P7 and adult BTBR mice. Adult BTBR mice were behaviorally analyzed for seizure susceptibility, sociability, communication deficits, and motor stereotypies, and monitored using chronic video-electroencephalography (EEG). Adult male and female BTBR mice treated at P7-P14 with LPS were more sensitive to pentylenetetrazol-induced seizures than saline-treated controls. ASD-like behaviors and hippocampal cytokine levels were unchanged between P7 LPS-treated BTBR mice and controls. EEG recordings from the dorsal hippocampus revealed a significant increase in number and frequency of seizures over the 4-week recording period (P60-P88) in BTBR mice postnatally treated with LPS at P7. These results indicate the presence of a comorbid epileptic phenotype in BTBR mice. These findings suggest that an early postnatal immune challenge can increase brain excitability in adult BTBR mice and reveal an underlying epilepsy phenotype. This novel animal model may enable the elucidation of specific molecular alterations that are associated with the concurrent presentation of ASD and epilepsy, which could facilitate the development of targeted therapies for individuals affected by this comorbidity.

Metabolic benefits of inhibition of p38α in white adipose tissue in obesity.

p38 has long been known as a central mediator of protein kinase A (PKA) signaling in brown adipocytes, which positively regulate the transcription of uncoupling protein 1 (UCP-1). However, the physiological role of p38 in adipose tissues, especially the white adipose tissue (WAT), is largely unknown. Here, we show that mice lacking p38α in adipose tissues display a lean phenotype, improved metabolism, and resistance to diet-induced obesity. Surprisingly, ablation of p38α causes minimal effects on brown adipose tissue (BAT) in adult mice, as evident from undetectable changes in UCP-1 expression, mitochondrial function, body temperature (BT), and energy expenditure. In contrast, genetic ablation of p38α in adipose tissues not only markedly facilitates the browning in WAT upon cold stress but also prevents diet-induced obesity. Consistently, pharmaceutical inhibition of p38α remarkably enhances the browning of WAT and has metabolic benefits. Furthermore, our data suggest that p38α deficiency promotes white-to-beige adipocyte reprogramming in a cell-autonomous manner. Mechanistically, inhibition of p38α stimulates the UCP-1 transcription through PKA and its downstream cAMP-response element binding protein (CREB), which form a positive feedback loop that functions to reinforce the white-to-beige phenotypic switch during cold exposure. Together, our study reveals that inhibition of p38α is able to promote WAT browning and confer metabolic benefits. Our study also indicates that p38α in WAT represents an exciting pharmacological target to combat obesity and metabolic diseases.

Use of homeobox gene expression patterns to determine anatomical regions of origin for body surface tissues derived from adult mice

Anatomical regions of the skin have distinct functions and anatomical characteristics, including thicker or thinner epidermis, more or fewer hair follicles, and lighter or darker skin. For a better therapeutic outcome of skin transplantation, site-specific characteristics of grafted tissues need to be taken into account in terms of their functionality and beauty. However, there is no method for evaluating positional information of epidermal cells. Homeobox genes are expressed along the anterior-posterior axis and direct the body plan in the animal development process. Although the expression of several HOX genes is known to be retained as the positional information in adult tissue, their expression patterns in the body surface tissues in adult mammals are still incompletely understood. In this study, we investigated the expression patterns of 40 homeobox genes, including 39 Hox genes and the paired box 6 (Pax6) gene, in body surface tissues of adult mice. On the basis of the results obtained, we proposed, for the first time, a method for determining anatomical regions of origin for body surface tissues derived from adult mice using Hox genes and Pax6. Evaluation of expression levels of at least 7 Hox genes and Pax6 should be sufficient to distinguish 11 anatomical body surface tissues derived from the adult mouse body. The proposed method may be useful not only for determining the origin of surface tissues from specific anatomical regions of the mammalian body but also for predicting positional information of epithelial cells generated from pluripotent stem cells.

Enriched expression of the ciliopathy gene Ick in cell proliferating regions of adult mice

Cilia are essential for sensory and motile functions across species. In humans, ciliary dysfunction causes “ciliopathies”, which show severe developmental abnormalities in various tissues. Several missense mutations in intestinal cell kinase (ICK) gene lead to endocrine-cerebro-osteodysplasia syndrome or short rib-polydactyly syndrome, lethal recessive developmental ciliopathies. We and others previously reported that Ick-deficient mice exhibit neonatal lethality with developmental defects. Mechanistically, Ick regulates intraflagellar transport and cilia length at ciliary tips. Although Ick plays important roles during mammalian development, roles of Ick at the adult stage are poorly understood. In the current study, we investigated the Ick gene expression in adult mouse tissues. RT-PCR analysis showed that Ick is ubiquitously expressed, with enrichment in the retina, brain, lung, intestine, and reproductive system. In the adult brain, we found that Ick expression is enriched in the walls of the lateral ventricle, in the rostral migratory stream of the olfactory bulb, and in the subgranular zone of the hippocampal dentate gyrus by in situ hybridization analysis. We also observed that Ick staining pattern is similar to pachytene spermatocyte to spermatid markers in the mature testis and to an intestinal stem cell marker in the adult small intestine. These results suggest that Ick is expressed in proliferating regions in the adult mouse brain, testis, and intestine.

HSF1 deficiency accelerates the transition from pressure overload-induced cardiac hypertrophy to heart failure through endothelial miR-195a-3p-mediated impairment of cardiac angiogenesis

Heat shock transcription factor 1 (HSF1) deficiency aggravates cardiac remodeling under pressure overload. However, the mechanism is still unknown. Here we employed microRNA array analysis of the heart tissue of HSF1-knockout (KO) mice to investigate the potential roles of microRNAs in pressure overload-induced cardiac remodeling under HSF-1 deficiency, and the profiles of 478 microRNAs expressed in the heart tissues of adult HSF1-KO mice were determined. We found that the expression of 5 microRNAs was over 2-fold higher expressed in heart tissues of HSF1-KO mice than in those of wild-type (WT) control mice. Of the overexpressed microRNAs, miR-195a-3p had the highest expression level in HSF1-null endothelial cells (ECs). Induction with miR-195a-3p in ECs significantly suppressed CD31 and VEGF, promoted AngII-induced EC apoptosis, and impaired capillary-like tube formation. In vivo, the upregulation of miR-195a-3p accentuated cardiac hypertrophy, increased the expression of β-MHC and ANP, and compromised systolic function in mice under pressure overload induced by transverse aortic constriction (TAC). By contrast, antagonism of miR-195a-3p had the opposite effect on HSF1-KO mice. Further experiments confirmed that AMPKα2 was the direct target of miR-195a-3p. AMPKα2 overexpression rescued the reduction of eNOS and VEGF, and the impairment of angiogenesis that was induced by miR-195a-3p. In addition, upregulation of AMPKα2 in the myocardium of HSF1-null mice by adenovirus-mediated gene delivery enhanced CD31, eNOS and VEGF, reduced β-MHC and ANP, alleviated pressure overload-mediated cardiac hypertrophy and restored cardiac function. Our findings revealed that the upregulation of miR-195a-3p due to HSF1 deficiency impaired cardiac angiogenesis by regulating AMPKα2/VEGF signaling, which disrupted the coordination between the myocardial blood supply and the adaptive hypertrophic response and accelerated the transition from cardiac hypertrophy to heart failure in response to pressure overload.

RNA/Protein Discordant Expression of Fndc5 in Central Nervous System Is Likely to Be Mediated Through microRNAs.

Fibronectin type III domain-containing 5 protein (Fndc5) is responsible for producing a secretory protein termed, "irisin." A modified expression of Fndc5 has been reported in different tissues during development, differentiation processes, as well as in metabolic events such as exercise. One of the important issues to be fixed is whether Fndc5 RNA level and protein content are concerted and modified hand in hand. Therefore, the aim of this study is to assess Fndc5 RNA and protein levels in various tissues of mouse and rat with emphasis on brain. Biopsies from various parts of neonatal and adult mouse and rat tissues were simultaneously assessed for transcript levels of Fndc5 and compared with the respective protein levels at the same time. Data indicated, unlike in muscle, no concerted fluctuations were observed for Fndc5 RNA and protein, especially in brain. Further look at four regions of brain (cerebellum, putamen, hippocampus, and cortex) revealed a similar discrepant expression. To hypothesize whether such discrepancy is arisen by miRNAs, we selected three main miRNAs, which were predicted to target Fndc5 and their expression levels were assessed in central nervous system (CNS) of mouse and hippocampus of rat. miRNA levels showed an antiparallel correlation with protein level of Fndc5, interpreting a putative role in regulating Fndc5 protein content in CNS. This phenomenon may represent the importance of governing Fndc5 content in neural cells, which seems to be crucial for neural function and differentiation.

Tissue specific differences in mitochondrial DNA maintenance and expression

The different cell types of multicellular organisms have specialized physiological requirements, affecting also their mitochondrial energy production and metabolism. The genome of mitochondria is essential for mitochondrial oxidative phosphorylation (OXHPOS) and thus plays a central role in many human mitochondrial pathologies. Disorders affecting mitochondrial DNA (mtDNA) maintenance are typically resulting in a tissue-specific pattern of mtDNA deletions and rearrangements. Despite this role in disease as well as a biomarker of mitochondrial biogenesis, the tissue-specific parameters of mitochondrial DNA maintenance have been virtually unexplored.In the presented study, we investigated mtDNA replication, topology, gene expression and damage in six different tissues of adult mice and sought to correlate these with the levels of known protein factors involved in mtDNA replication and transcription. Our results show that while liver and kidney cells replicate their mtDNA using the asynchronous mechanism known from cultured cells, tissues with high OXPHOS activity, such as heart, brain, skeletal muscle and brown fat, employ a strand-coupled replication mode, combined with increased levels of recombination. The strand-coupled replication mode correlated also with mtDNA damage levels, indicating that the replication mechanism represents a tissue-specific strategy to deal with intrinsic oxidative stress. While the preferred replication mode did not correlate with mtDNA transcription or the levels of most known mtDNA maintenance proteins, mtSSB was most abundant in tissues using strand-asynchronous mechanism. Although mitochondrial transcripts were most abundant in tissues with high metabolic rate, the mtDNA copy number per tissue mass was remarkably similar in all tissues. We propose that the tissue-specific features of mtDNA maintenance are primarily driven by the intrinsic reactive oxygen species exposure, mediated by DNA repair factors, whose identity remains to be elucidated.

Dissecting Cell-Type Composition and Activity-Dependent Transcriptional State in Mammalian Brains by Massively Parallel Single-Nucleus RNA-Seq

Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues such as adult mammalian brains is challenging. Here, we integrate sucrose-gradient-assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-seq approach (sNucDrop-seq), which is free of enzymatic dissociation and nucleus sorting. By profiling ∼18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, invivo.

Wnt7a induces a unique phenotype of monocyte-derived macrophages with lower phagocytic capacity and differential expression of pro- and anti-inflammatory cytokines.

The variation of macrophage functions suggests the involvement of multiple signalling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or 'danger' signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact on monocyte differentiation is not clear. In this study, we assessed the role of Wnt1 and Wnt7a on the differentiation of monocytes and the subsequent phenotype and function of monocyte-derived macrophages (MDMs). We show that Wnt7a decreased the expression of CD14, CD11b, CD163 and CD206, whereas Wnt1 had no effect. The Wnt7a effect on CD11b was also observed in the brain and spleen of Wnt7a-/- adult brain mouse tissue and in embryonic Wnt7a-/- tissue. Wnt7a reduced the phagocytic capacity of M-MDMs, decreased interleukin-10 (IL-10) and IL-12 secretion and increased IL-6 secretion. Collectively, these findings demonstrate that Wnt7a generates an MDM phenotype with both pro-inflammatory and alternative MDM cytokine profiles and reduced phagocytic capacity. As such, Wnt7a can have a significant impact on macrophage responses in health and disease.

Osteoclast stimulation factor 1 (Ostf1) KNOCKOUT increases trabecular bone mass in mice

Osteoclast stimulation factor 1 (OSTF1) is an SH3-domain containing protein that was initially identified as a factor involved in the indirect activation of osteoclasts. It has been linked to spinal muscular atrophy in humans through its interaction with SMN1, and is one of six genes deleted in a human developmental microdeletion syndrome. To investigate the function of OSTF1, we generated an Ostf1 knockout mouse model, with exons 3 and 4 of Ostf1 replaced by a LacZ orf. Extensive X-Gal staining was performed to examine the developmental and adult expression pattern, followed by phenotyping. We show widespread expression of the gene in the vasculature of most organs and in a number of cell types in adult and embryonic mouse tissues. Furthermore, whilst SHIRPA testing revealed no behavioural defects, we demonstrate increased trabecular mass in the long bones, confirming a role for OSTF1 in bone development.

Enriched environment improves behavioral performance and attenuates inflammatory response induced by TNF-α in healthy adult mice

Several studies have demonstrated the neuroprotective effect of enriched environment (EE) and its positive effect on cognitive performance in pathological conditions, such as neurodegenerative diseases, epilepsy, and traumatic brain injury. However, the immunomodulatory effect of EE in normal rodents is not well characterized. To assess the immunomodulatory effect of EE, we randomly assigned normal mice to EE housing or standard environmental (SE) housing for 3 weeks. Behavioral alterations were evaluated by open field, fear conditioning, and Morris water maze tests. Immunohistochemical staining was performed to assess the expression of behavioral-related proteins, and enzyme-linked immunosorbent assay (ELISA) for brain-derived neurotrophic factor (BDNF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) was also performed. We also measured the levels of RIP1 and RIP3 proteins using western blotting. EE significantly improved the cognitive performance which was associated with the increased expressions of BDNF, ionized calcium-binding adapter molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP); EE did not influence any morphological changes in the brain tissue in adult mice; however, increased resistance to inflammation induced by TNF-α was observed. These findings indicate that EE can positively influence cognitive and behavioral performance in healthy adult mice by exerting environ-immuno effect on neural function.

Effects of a High-Fat Diet on Adipose Tissue CD8+ T Cells in Young vs. Adult Mice.

T cells are involved in chronic inflammation of adipose tissue in obese conditions. However, the impact of age on the adipose T cells remains unknown. In this study, we investigated T cells in the white adipose tissue of young and adult mice. Obesity was induced in the mice using a high-fat diet (HFD) for 14weeks. The young mice were fed an HFD at 3weeks old, and adult mice were fed the HFD at 12weeks old. Relative to adult mice, the young mice gained less fat and exhibited better glucose tolerance. Their adipose tissue contained more CD8+ T cells and higher levels of pro-inflammatory cytokines. Young mice showed a larger increase in CD4+ T cells. The young and adult mice showed similar insulin tolerance. HFD reduced the colon muscle layer, which was more obvious in the young mice. These data suggested that young and adult mice exhibit different responses to an HFD in terms of adipose tissue, glucose tolerance, and the colon muscle layer. The increase in CD8+ T cells and CD4+ T cells, together with higher levels of pro-inflammatory cytokines, suggested elevated inflammation in the presence of less fat gain in the young mice, which was unexpected. The significance of this inflammation remains unknown. We propose that inflammation might inhibit energy storage in the adipose tissue to provide more energy to the lean body mass in favor of growth in the young mice. The present study provides another example of the beneficial effect of inflammation in physiological conditions.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Supramolecular structure of dietary fat in early life modulates expression of markers for mitochondrial content and capacity in adipose tissue of adult mice

BackgroundPrevious studies have shown that early life nutrition can modulate the development of white adipose tissue and thereby affect the risk on obesity and metabolic disease later in life. For instance, postnatal feeding with a concept infant milk formula with large, phospholipid coated lipid droplets (Concept, Nuturis®), resulted in reduced adiposity in adult mice. The present study investigated whether differences in cell energy metabolism, using markers of mitochondrial content and capacity, may contribute to the observed effects.MethodsC57Bl/6j male mice were exposed to a rodent diet containing the Concept (Concept) or standard (CTRL) infant milk formula from postnatal day 16 until postnatal day 42, followed by a western style diet challenge until postnatal day 98. Markers for mitochondrial content and capacity were analyzed in retroperitoneal white adipose tissue and gene expression of metabolic markers was measured in both retroperitoneal white adipose tissue and muscle tibialis (M. tibialis) at postnatal day 98.ResultsIn retroperitoneal white adipose tissue, the Concept group showed higher citrate synthase activity and mitochondrial DNA expression compared to the CTRL group (p < 0.05). In addition, protein expression of mitochondrial cytochrome c oxidase subunit I of the oxidative phosphorylation pathway/cascade was increased in the Concept group compared to CTRL (p < 0.05). In the M. tibialis, gene expression of uncoupling protein 3 was higher in the Concept compared to the CTRL group. Other gene and protein expression markers for mitochondrial oxidative capacity were not different between groups.ConclusionPostnatal feeding with large, phospholipid coated lipid droplets generating a different supramolecular structure of dietary lipids enhances adult gene and protein expression of specific mitochondrial oxidative capacity markers, indicative of increased substrate oxidation in white adipose tissue and skeletal muscle. Although functional mitochondrial capacity was not measured, these results may suggest that adaptations in mitochondrial function via early feeding with a more physiological structure of dietary lipids, could underlie the observed beneficial effects on later life adiposity.

An ex vivo spinal cord injury model to study ependymal cells in adult mouse tissue

Traumatic spinal cord injury is characterized by an initial cell loss that is followed by a concerted cellular response in an attempt to restore the damaged tissue. Nevertheless, little is known about the signaling mechanisms governing the cellular response to injury. Here, we have established an adult ex vivo system that exhibits multiple hallmarks of spinal cord injury and allows the study of complex processes that are difficult to address using animal models. We have characterized the ependymal cell response to injury in this model system and found that ependymal cells can become activated, proliferate, migrate out of the central canal lining and differentiate in a manner resembling the in vivo situation. Moreover, we show that these cells respond to external adenosine triphosphate and exhibit spontaneous Ca2+ activity, processes that may play a significant role in the regulation of their response to spinal cord injury. This model provides an attractive tool to deepen our understanding of the ependymal cell response after spinal cord injury, which may contribute to the development of new treatment options for spinal cord injury.

ROLE OF WHEAT GERM OIL AGAINST CARBON TETRACHLORIDE-INDUCED NEUROTOXICITY IN BRAIN TISSUES OF ADULT MALE MICE

The present study aimed to evaluate the possible curative effect of wheat germ oil (WGO) against carbon tetrachloride (CCl4)-induced neurotoxicity accompanied to the oxidative stress in brain tissue of adult male mice. A total of 24 mice were divided into 4 groups, 6 animals for each group. Control group: received oral corn oil for 10 days (0.5 mL/kg body weight). Wheat germ oil (WGO) group: received corn oil (0.5 mL/kg body weight) orally for 2 days then WGO (1400 mg/kg body weight) for 8 days. Carbon tetrachloride (CCl4) group: received CCl4 in corn oil orally (0.5 mL/kg body weight) for 2 days then received corn oil (0.5 mL/kg body weight) for 8 days. WGO+ CCl4 group: received CCl4 in corn oil (0.5 mL/kg body weight) orally for 2 days then WGO (1400 mg/kg body weight) for 8 days. Administration of CCl4 induced increment in malondialdehyde (MDA), nitric oxide (NO), oxidized glutathione (GSSG), 8-hydroxy-2-deoxyguanosine (8-OHdG), adenosine di- and mono-phosphates (ADP & AMP), while caused drop in reduced glutathione (GSH), adenosine triphosphate (ATP) and monoamines in brain tissues. Administration of WGO could reduce neurotoxicity and mitochondrial dysfunction caused by CCl4. WGO exhibited curative effect in restoring energy production, improving oxidative status, reducing DNA degradation and modulating neurotransmitters levels. In conclusion, CCl4-induced neurotoxicity aggravated the oxidative stress in brain tissues, causing DNA fragmentation, inhibited energy production and suppressed monoamines release. One the other hand, administration of WGO attained improvement in the aforementioned negative changes.

Impairment of insulin signalling in peripheral tissue fails to extend murine lifespan.

SummaryImpaired insulin/IGF1 signalling has been shown to extend lifespan in model organisms ranging from yeast to mammals. Here we sought to determine the effect of targeted disruption of the insulin receptor (IR) in non‐neuronal tissues of adult mice on the lifespan. We induced hemizygous (PerIRKO +/−) or homozygous (PerIRKO −/−) disruption of the IR in peripheral tissue of 15‐weeks‐old mice using a tamoxifen‐inducible Cre transgenic mouse with only peripheral tissue expression, and subsequently monitored glucose metabolism, insulin signalling and spontaneous death rates over 4 years. Complete peripheral IR disruption resulted in a diabetic phenotype with increased blood glucose and plasma insulin levels in young mice. Although blood glucose levels returned to normal, and fat mass was reduced in aged PerIRKO −/− mice, their lifespan was reduced. By contrast, heterozygous disruption had no effect on lifespan. This was despite young male PerIRKO +/− mice showing reduced fat mass and mild increase in hepatic insulin sensitivity. In conflict with findings in metazoans like Caenorhabditis elegans and Drosophila melanogaster, our results suggest that heterozygous impairment of the insulin signalling limited to peripheral tissues of adult mice fails to extend lifespan despite increased systemic insulin sensitivity, while homozygous impairment shortens lifespan.

CRISPR/Cas9 – Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah−/− liver failure mice, offering an efficient approach for precisely targeted gene therapies.

Dynamic silencing of somatic L1 retrotransposon insertions reflects the developmental and cellular contexts of their genomic integration

BackgroundThe ongoing mobilization of mammalian transposable elements (TEs) contributes to natural genetic variation. To survey the epigenetic control and expression of reporter genes inserted by L1 retrotransposition in diverse cellular and genomic contexts, we engineered highly sensitive, real-time L1 retrotransposon reporter constructs.ResultsHere we describe different patterns of expression and epigenetic controls of newly inserted sequences retrotransposed by L1 in various somatic cells and tissues including cultured human cancer cells, mouse embryonic stem cells, and tissues of pseudofounder transgenic mice and their progeny. In cancer cell lines, the newly inserted sequences typically underwent rapid transcriptional gene silencing, but they lacked cytosine methylation even after many cell divisions. L1 reporter expression was reversible and oscillated frequently. Silenced or variegated reporter expression was strongly and uniformly reactivated by treatment with inhibitors of histone deacetylation, revealing the mechanism for their silencing. By contrast, de novo integrants retrotransposed by L1 in pluripotent mouse embryonic stem (ES) cells underwent rapid silencing by dense cytosine methylation. Similarly, de novo cytosine methylation also was identified at new integrants when studied in several distinct somatic tissues of adult founder mice. Pre-existing L1 elements in cultured human cancer cells were stably silenced by dense cytosine methylation, whereas their transcription modestly increased when cytosine methylation was experimentally reduced in cells lacking DNA methyltransferases DNMT1 and DNMT3b. As a control, reporter genes mobilized by piggyBac (PB), a DNA transposon, revealed relatively stable and robust expression without apparent silencing in both cultured cancer cells and ES cells.ConclusionsWe hypothesize that the de novo methylation marks at newly inserted sequences retrotransposed by L1 in early pre-implantation development are maintained or re-established in adult somatic tissues. By contrast, histone deacetylation reversibly silences L1 reporter insertions that had mobilized at later timepoints in somatic development and differentiation, e.g., in cancer cell lines. We conclude that the cellular contexts of L1 retrotransposition can determine expression or silencing of newly integrated sequences. We propose a model whereby reporter expression from somatic TE insertions reflects the timing, molecular mechanism, epigenetic controls and the genomic, cellular and developmental contexts of their integration.