The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an ...efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.
Rodents' ultrasonic vocalizations (USVs) provide useful information for assessing their social behaviors. Despite previous efforts in classifying subcategories of time-frequency patterns of USV ...syllables to study their functional relevance, methods for detecting vocal elements from continuously recorded data have remained sub-optimal. Here, we propose a novel procedure for detecting USV segments in continuous sound data containing background noise recorded during the observation of social behavior. The proposed procedure utilizes a stable version of the sound spectrogram and additional signal processing for better separation of vocal signals by reducing the variation of the background noise. Our procedure also provides precise time tracking of spectral peaks within each syllable. We demonstrated that this procedure can be applied to a variety of USVs obtained from several rodent species. Performance tests showed this method had greater accuracy in detecting USV syllables than conventional detection methods.
The rise of multi-drug-resistant (MDR) bacteria has spurred renewed interest in the use of bacteriophages in therapy. However, mechanisms contributing to phage-mediated bacterial clearance in an ...animal host remain unclear. We investigated the effects of host immunity on the efficacy of phage therapy for acute pneumonia caused by MDR Pseudomonas aeruginosa in a mouse model. Comparing efficacies of phage-curative and prophylactic treatments in healthy immunocompetent, MyD88-deficient, lymphocyte-deficient, and neutrophil-depleted murine hosts revealed that neutrophil-phage synergy is essential for the resolution of pneumonia. Population modeling of in vivo results further showed that neutrophils are required to control both phage-sensitive and emergent phage-resistant variants to clear infection. This “immunophage synergy” contrasts with the paradigm that phage therapy success is largely due to bacterial permissiveness to phage killing. Lastly, therapeutic phages were not cleared by pulmonary immune effector cells and were immunologically well tolerated by lung tissues.
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•Efficacious phage therapy to pulmonary P. aeruginosa requires innate immune components•Neutrophils are required to control phage-sensitive and emergent phage-resistant bacteria•Models predict “immunophage synergy” arises due to nonlinear feedback
The mechanisms underlying phage-mediated bacterial clearance in an animal host remain unclear. By coupling animal experiments and in silico modeling, Roach et al. show that host innate immunity is essential for the efficacy of phages in treating respiratory bacterial infections, defining the concept of “immunophage synergy.”
A vast number of different neuronal activity patterns could each induce a different set of activity-regulated genes. Mapping this coupling between activity pattern and gene induction would allow ...inference of a neuron’s activity-pattern history from its gene expression and improve our understanding of activity-pattern-dependent synaptic plasticity. In genome-scale experiments comparing brief and sustained activity patterns, we reveal that activity-duration history can be inferred from gene expression profiles. Brief activity selectively induces a small subset of the activity-regulated gene program that corresponds to the first of three temporal waves of genes induced by sustained activity. Induction of these first-wave genes is mechanistically distinct from that of the later waves because it requires MAPK/ERK signaling but does not require de novo translation. Thus, the same mechanisms that establish the multi-wave temporal structure of gene induction also enable different gene sets to be induced by different activity durations.
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•Distinct durations of neuronal activity induce different gene expression profiles•Neuronal activity history can be inferred from gene expression•Brief activity induces a gene set defined by sensitivity to MAPK/ERK signaling•H3K27ac and eRNA induction are two separable steps of enhancer activation
Tyssowski et al. report that different durations of neuronal activity induce different gene expression profiles, enabling inference of past neuronal activity from gene expression data. Furthermore, they show that MAPK/ERK signaling partially establishes this activity-pattern-to-gene-induction coupling.
Obesity develops when energy intake exceeds energy expenditure. Promoting brown adipose tissue formation and function increases energy expenditure and hence may counteract obesity. Berberine (BBR) is ...a compound derived from the Chinese medicinal plant Coptis chinensis. Here we show that BBR increases energy expenditure, limits weight gain, improves cold tolerance and enhances brown adipose tissue (BAT) activity in obese db/db mice. BBR markedly induces the development of brown-like adipocytes in inguinal, but not epididymal adipose depots. BBR also increases expression of UCP1 and other thermogenic genes in white and BAT and primary adipocytes via a mechanism involving AMPK and PGC-1α. BBR treatment also inhibits AMPK activity in the hypothalamus, but genetic activation of AMPK in the ventromedial nucleus of the hypothalamus does not prevent BBR-induced weight loss and activation of the thermogenic programme. Our findings establish a role for BBR in regulating organismal energy balance, which may have potential therapeutic implications for the treatment of obesity.
Salt-sensitive hypertension is a major risk factor for cardiovascular disorders. Our previous proteomic study revealed substantial differences in several proteins between Dahl salt-sensitive (SS) ...rats and salt-insensitive consomic SS.13(BN) rats. Subsequent experiments indicated a role of fumarase insufficiency in the development of hypertension in SS rats. In the present study, a global metabolic profiling study was performed using gas chromatography/mass spectrometry (GC/MS) in plasma of SS rats (n=9) and SS.13(BN) rats (n=8) on 0.4% NaCl diet, designed to gain further insights into the relationship between alterations in cellular intermediary metabolism and predisposition to hypertension. Principal component analysis of the data sets revealed a clear clustering and separation of metabolic profiles between SS rats and SS.13(BN) rats. 23 differential metabolites were identified (P<0.05). Higher levels of five TCA cycle metabolites, fumarate, cis-aconitate, isocitrate, citrate and succinate, were observed in SS rats. Pyruvate, which connects TCA cycle and glycolysis, was also increased in SS rats. Moreover, lower activity levels of fumarase, aconitase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase were detected in the heart, liver or skeletal muscles of SS rats. The distinct metabolic features in SS and SS.13(BN) rats indicate abnormalities of TCA cycle in SS rats, which may play a role in predisposing SS rats to developing salt-sensitive hypertension.
After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, ...some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.
Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to ...activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.
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•RIPK1 and RIPK3 kinases promote TRIF-dependent cytokine production by LPS in vitro•RIPK1 and RIPK3 kinase-dependent inflammation is independent of cell death•Erk1/2 mediates RIPK1 and RIPK3 kinase dependent inflammation•RIPK1 kinase and RIPK3 are mediators of LPS-induced cytokine production in vivo
Kinase activities of RIPK1 and RIPK3 are critical for necroptotic cell death. Degterev and colleagues demonstrate that RIPK1 and RIPK3 kinases also direct inflammatory gene expression induced by Toll-like receptor 4 ligand, lipopolysaccharide, in vitro, and in vivo. This regulation is independent of necroptosis and requires Erk1/2, cFos, and NF-κB.
To identify highly informative markers for a large number of commonly employed murine crosses, we selected a subset of the extant mouse simple sequence length polymorphism (SSLP) marker set for ...further development. Primer pairs for 314 SSLP markers were designed and typed against 54 inbred mouse strains. We designed new PCR primer sequences for the markers selected for multiplexing using the fluorescent dyes FAM, VIC, NED, and ROX. The number of informative markers for C57BL/6J x DBA/2J is 217, with an average spacing of 6.8 centiMorgans (cM). For all other pairs of strains, the mean number of informative markers per cross is 197.0 (SD 37.8) with a mean distance between markers of 6.8 cM (SD 1.1). To confirm map positions of the 224 markers in our set that are polymorphic between Mus musculus and Mus spretus, we used The Jackson Laboratory (TJL) interspecific backcross mapping panel (TJL BSS); 168 (75%) of these markers had not been previously mapped in this cross by other investigators, adding new information to this community map resource. With this large data set, we sought to reconstruct a phylogenetic history of the laboratory mouse using Wagner parsimony analysis. Our results are largely congruent with the known history of inbred mouse strains.
Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled ...muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML.
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