Biology subjects

Gu, B.

Publications and source records attributed to Gu, B..

50 records · Page 3Linked to original sources

bioRxiv2022

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues. HighlightsO_LIAuxin-inducible degradation of endogenously tagged proteins in living mice and a range of primary cells. C_LIO_LIMost but not all cell types are competent for degradation C_LIO_LIDosage of the tagged protein, E3 ligase substrate receptor and ligand can all determine degradation kinetics C_LIO_LIRapid degradation of condensin subunits in lymphocytes reveals stage-specific requirements during cell division C_LI

genetics↗

bioRxiv2022

The generation of a Nutm1 knock-in reporter mouse line for imaging post-meiotic spermatogenesis

Spermiogenesis, the post-meiotic stage of sperm development, is critical for normal male fertility. Many genetic defects and environmental assaults that affect spermiogenesis have been shown to be associated with male infertility. In addition, this later stage of spermatogenesis has been proposed to be an ideal target for male contraceptive development. The mouse is a widely used model for studying the mechanisms of spermatogenesis and spermiogenesis. However, due to the complexity and the asynchronous nature of spermatogenesis in adult testis, it is challenging to study molecular processes restricted to this specific developmental stage. It is also challenging to monitor the spermiogenesic activity in live mice, which is critical for screening for fertility-modulating interventions such as contraceptives. Here we reported the development of a Nutm1-T2A-luciferase 2(Luc2)-tandem Tomato(TdTomato) knock-in reporter mouse model that specifically labels post-meiotic spermatids. Homozygous reporter mice are healthy and fully fertile, demonstrating no interference with the normal functions of the Nutm1 gene by the reporter. We demonstrated the visualization of post-meiotic spermatids by fluorescent imaging of the TdTomato reporter in both live and fixed testis tissues. We also demonstrated bioluminescence imaging of Nutm1 expressing cells in live mice. The Nutm1-T2A-Luc2TdTomato reporter mouse can serve as a valuable tool for studying spermiogenesis.

developmental biology↗

bioRxiv2021

Live imaging YAP signaling in vivo with fusion reporter mice

YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATs kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behavior of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signaling in vivo in many different situations.

developmental biology↗

bioRxiv2021

ZFP541 is indispensable for pachytene progression by interacting with KCTD19 and activates meiotic gene expression in mouse spermatogenesis

Meiosis is essential for fertility in sexually reproducing species, extensive studies tried to delineate this sophisticated process. Notwithstanding, the molecules involved in meiosis have not been fully characterized. In this study, we investigate the role of zinc finger protein 541 (ZFP541) and its interacting protein potassium channel tetramerization domain containing 19 (KCTD19) in mice. We demonstrate that they are indispensable for male fertility by regulating proper pachytene progression. ZFP541 is expressed starting from leptotene to round spermatids, and KCTD19 is initially expressed in pachytene. Depletion of Zfp541 or Kctd19 leads to infertility in male mice, and exhibits retarded progression from early to mid/late pachynema. In addition, Zfp541-/- spermatocytes show abnormal programmed DNA double-strand breaks (DSBs) repair, impaired crossover formation/resolution, and asynapsis of the XY chromosomes. Immunoprecipitation-mass spectrometry (IP-MS) and in vitro Co-IP reveal that ZFP541 interacts with KCTD19, histone deacetylase 1/2 (HDAC1), HDAC2 and deoxynucleotidyltransferase terminal-interacting protein 1 (DNTTIP1). Furthermore, RNA-seq and CUT&Tag analyses demonstrate that ZFP541 binds to the promoter regions of genes involved in meiosis and post-meiosis including Kctd19, and activates their transcription. Taken together, our studies reveal a ZFP541-Kctd19 transcription regulatory axis and the crucial role of ZFP541 and KCTD19 for pachytene progression and fertility in male mice.

developmental biology↗

bioRxiv2020

Selection of internal references for transcriptomics and RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines

Transcriptomics has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the most qualified reference genes for the relative quantitation analysis of transcriptomics and real-time quantitative reverse-transcription PCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most used internal reference gene, was significantly unstable. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. Our recommended internal reference genes may improve the accuracy and reproducibility of the results of transcriptomics and real-time quantitative reverse-transcription PCR in further gene expression analyses of NF1 related tumors.

cancer biology↗

bioRxiv2020

Content and performance of the MiniMUGA genotyping array, a new tool to improve rigor and reproducibility in mouse research

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well annotated genome, wealth of genetic resources and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost effective, informative and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole genome sequencing. Here we describe the content and performance of a new Mouse Universal Genotyping Array (MUGA). MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: 1) chromosomal sex determination, 2) discrimination between substrains from multiple commercial vendors, 3) diagnostic SNPs for popular laboratory strains, 4) detection of constructs used in genetically engineered mice, and 5) an easy to interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA we genotyped 6,899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived and recombinant inbred lines. Here we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, the extension of the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. There is preliminary but striking evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the main stock for a significant action of the genotyped inbred samples. We conclude that MiniMUGA is a valuable platform for genetic QC and important new tool to the increase rigor and reproducibility of mouse research.

genetics↗

bioRxiv2019

QTL-Seq Identifies QTL for Relative Electrical Conductivity Associated with Heat Tolerance in Bottle Gourd (Lagenaria Siceraria)

Heat is a major abiotic stressor seriously affects watermelon produce; however, its effects may be mitigated through grafting onto heat tolerant bottle gourd rootstock. To understand the genetic basis and select reliable markers are necessary in the variety breeding process. In this paper, the relative electric conductivity (REC) was used as a visual indicator for heat stress to study the genetics and SNP marker of heat tolerance in bottle gourd via QTL-seq approach. The results showed that recessive inheritance and a major QTL locus controlled REC related to heat tolerance. Seven heat-tolerant loci (qHT1.1, qHT2.1, qHT2.2, qHT5.1, qHT6.1, qHT7.1, and qHT8.1) exhibited high {Delta}(SNP-index) values (ranging from 0.19-0.38), and of these, the greatest value (0.32, P < 0.01) and greatest number of detected SNPs (9052) were found in chromosome 2 at qHT2.1 region (11.03 - 19.25 Mb); qHT2.1was taken as a promising major QTL for heat tolerance in bottle gourd. A total of 34 putative candidate genes related to heat stress were detected within the qHT2.1 region. Three polymorphic SNPs (BG_GLEAN_10022642, BG_GLEAN_10022727, and BG_GLEAN_10022589) that are involved in pollen sterility, intracellular transport, and signal recognition were validated and exhibited significant marker-trait association that could be used in heat tolerant molecular breeding for bottle gourd. The qHT2.1region is an important finding can be used for fine mapping and discovering new genes associated with heat tolerance in bottle gourd.

molecular biology↗

bioRxiv2019

Collaborative Cross Mouse Populations as a Resource for the Study of Epilepsy

Epilepsy is a neurological disorder with complex etiologies and genetic architecture. Animal models have a critical role in understanding the pathophysiology of epilepsy. Here we studied epilepsy utilizing a genetic reference population of Collaborative Cross (CC) mice with publicly available whole genome sequences. We measured multiple epilepsy traits in 35 CC strains, and we identified novel animal models that exhibit extreme outcomes in seizure susceptibility, seizure propagation, epileptogenesis, and sudden unexpected death in epilepsy. We performed QTL mapping in an F2 population and identified seven novel and one previously identified loci associated with seizure sensitivity. We combined whole genome sequence and hippocampal gene expression to pinpoint biologically plausible candidate genes and candidate variants associated with seizure sensitivity. These resources provide a powerful toolbox for studying complex features of seizures and for identifying genes associated with particular seizure outcomes, and hence will facilitate the development of new therapeutic targets for epilepsy.

neuroscience↗

bioRxiv2019

Cannabidiol attenuates seizures and EEG abnormalities in Angelman syndrome model mice

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by intellectual disability, lack of speech, ataxia, EEG abnormalities, and epilepsy. Seizures in AS individuals are often refractory to existing antiepileptic medications. Therefore, there is an unmet need for better seizure control, which could potentially improve other symptomatic domains such as cognitive function. Cannabidiol (CBD), a major phytocannabinoid constituent of cannabis, has anti-seizure activity and behavioral benefits in preclinical and clinical studies for some disorders associated with epilepsy, suggesting that the same could be true for AS. Here we show that acute CBD (100 mg/kg) attenuated hyperthermia- and acoustically-induced seizures in a mouse model of AS. However, neither acute CBD nor a two-weeklong course of CBD administered immediately after a kindling protocol could halt the pro-epileptogenic plasticity observed in AS model mice. CBD had a mild sedative effect, but did not have a major impact on motor performance. CBD abrogated the enhanced delta rhythms observed in AS model mice, indicating that CBD administration could also help normalize the EEG deficits observed in individuals with AS. Our results provide critical preclinical evidence supporting CBD treatment of seizures and alleviation of EEG abnormalities in AS, and will thus help guide the rational development of CBD as an AS adjunctive treatment.

pharmacology and toxicology↗

bioRxiv2019

Comparative genome analysis of 12 Shigella sonnei strains: virulence, resistance, and their interactions

Shigellosis is a highly infectious disease that are mainly transmitted via faecal-oral contact of the bacteria Shigella. Four species have been identified in Shigella genus, among which S. flexneri is used to be the most prevalent species globally and commonly isolated from developing countries. However, it is being replaced by S. sonnei that is currently the main causative agent for dysentery pandemic in many emerging industrialized countries such as Asia and the Middle East with unclear reasons. For a better understanding of S. sonnei virulence and antibiotic resistance, we sequenced 12 clinical S. sonnei strains with varied antibiotic-resistance profiles collected from four cities in Jiangsu Province, China. Phylogenomic analysis clustered antibiotic sensitive and resistant S. sonnei into two distinct groups while pangenome analysis reveals the presence and absence of featured genes in each group. Screening of 31 classes of virulence factors found out that type 2 secretion system is doubled in resistant strains. Further principle component analysis based on the interactions between virulence and resistance indicated that abundant virulence factors are associated with higher resistant phenotypes. The result present here is based on statistical analysis of a small sample size and serves basically as a guidance for further experimental and theoretical studies.

microbiology↗

bioRxiv2018

Super-enhancer impairment is a link between MLL4 inactivated lung tumors and their vulnerability to glycolysis pathway inhibition

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

cancer biology↗

bioRxiv2018

YggG is a Novel SPI-1 Effector Essential for Salmonella Virulence

Salmonella is a leading cause of food borne illness and poses a major public health problem worldwide. Two type III secretion systems (TTSSs) are responsible for the delivery of a series of bacterial effectors into the infected cells to reprogram host cell functions to promote bacterial invasion and intracellular survival/replication. Nearly half of Salmonella genes encode proteins that are annotated as \"hypothetical\". We hypothesize that some of these hypothetical proteins might be TTSS effectors and participate in Salmonella virulence. In this study, we employed an in silico screen to identify putative TTSS effector proteins and performed a large scale screening of Salmonella effectors using a {beta}-lactamase protein translocation reporter assay. We identified 22 novel effectors that have not been previously reported. One of the effector - YggG, is found to be cytotoxic to mammalian cells when overexpressed suggesting interference with mammalian cell functions and survival. Importantly, Salmonella strains lacking YggG has reduced virulence in the mouse infection model. Our study demonstrated that YggG and its protease activity are required for Salmonella virulence in the mouse infection model. Surprisingly, no detectable roles in invasion into epithelial cells and no effect on the survival and replication inside cultured macrophages. We speculate that YggG may be involved in altering some aspects of the host immune system to promote infection. Our finding significantly expands the number of known Salmonella effectors, and laid a solid foundation in further understanding Salmonella pathogenesis.\n\nAbstract ImportanceSalmonellosis continues to be public health concern in the USA and the world. Salmonella delivers a set of bacterial proteins (effectors) into the host cells to invade non-phagocytic cells and to replicate inside the infected cells. Identification and characterization of these effectors are pivotal to the understanding of how Salmonella causes diseases. In addition, Nearly half of Salmonella genes encode proteins that are annotated as \"hypothetical\". We have identified 22 novel effectors that have not been previously reported. Our finding significantly expands the number of known Salmonella effectors, and laid a solid foundation in further understanding Salmonella pathogenesis.

microbiology↗

bioRxiv2017

Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

Rapid and efficient generation of large fragment targeted knock-in mouse models is still a major hurdle in mouse genetics. Here we developed 2C-HR-CRISPR, a highly efficient gene editing method based on introducing CRISPR reagents into mouse embryos at the 2-cell stage, taking advantage of the likely increase in HR efficiency during the long G2 phase and open chromatin structure of the 2-cell embryo. With 2C-HR-CRISPR and a modified biotin-streptavidin approach to localize repair templates to target sites, we rapidly targeted 20 endogenous genes that are expressed in mouse blastocysts with fluorescent reporters and generated reporter mouse lines. We showcase the first live triple-color blastocyst with all three lineages differentially reported. Additionally, we demonstrated efficient double targeting, enabling rapid assessment of the auxin-inducible degradation system for probing protein function in mouse embryos. These methods open up exciting avenues for exploring cell fate decisions in the blastocyst and later stages of development. We also suggest that 2C-HR-CRISPR can be a better alternative to random transgenesis by ensuring transgene insertions at defined safe harbor sites.

genetics↗

bioRxiv2017

Carbon pool trends and dynamics within a subtropical peatland during long-term restoration

BackgroundThe Florida Everglades has undergone significant ecological change spanning the continuum of disturbance to restoration. While the restoration effort is not complete and the ecosystem continues to experience short duration perturbations, a better understanding of long-term C dynamics of the Everglades is needed to facilitate new restoration efforts. This study evaluated temporal trends of different aquatic carbon (C) pools of the northern Everglades Protection Area over a 20-year period to gauge historic C cycling patterns. Dissolved inorganic C (DIC), dissolved organic C (DOC), particulate organic C (POC), and surface water carbon dioxide (pCO2(aq)) were investigated between May 1, 1994 and April 30, 2015.\n\nResultsAnnual mean concentrations of DIC, DOC, POC, and pCO2(aq) significantly decreased through time or remained constant across the Water Conservation Areas (WCAs). Overall, the magnitude of the different C pools in the water column significantly differed between regions. Outgassing of CO2 was dynamic across the Everglades ranging from 420 to 2001 kg CO2 yr-1. Overall the historic trend in CO2 flux from the marsh declined across our study area while pCO2(aq) largely remained somewhat constant with the exception of Water Conservation Area 2 which experienced significant declines in pCO2(aq). Particulate OC concentrations were consistent between WCAs, but a significantly decreasing trend in annual POC concentrations were observed.\n\nConclusionsHydrologic condition and nutrient inputs significantly influenced the balance, speciation, and flux of C pools across WCAs suggesting a subsidy-stress response in C dynamics relative to landscape scale responses in nutrient availability. The interplay between nutrient inputs and hydrologic condition exert a driving force on the balance between DIC and DOC production via the metabolism of organic matter which forms the base of the aquatic foodweb. Along the restoration trajectory as water quality and hydrology continues to improve it is expected that C pools will respond accordingly.

ecology↗