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Loss of HCN2 leads to delayed gastrointestinal motility and reduced energy intake in mice.
Fisher DW, Luu P, Agarwal N, Kurz JE, Chetkovich DM
(2018) PLoS One 13: e0193012
MeSH Terms: Animals, Blood Glucose, Energy Intake, Female, Gastrointestinal Motility, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutagenesis, Insertional, Sequence Analysis, DNA
Show Abstract · Added April 2, 2019
Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are important regulators of excitability in neural, cardiac, and other pacemaking cells, which are often altered in disease. In mice, loss of HCN2 leads to cardiac dysrhythmias, persistent spike-wave discharges similar to those seen in absence epilepsy, ataxia, tremor, reduced neuropathic and inflammatory pain, antidepressant-like behavior, infertility, and severely restricted growth. While many of these phenotypes have tissue-specific mechanisms, the cause of restricted growth in HCN2 knockout animals remains unknown. Here, we characterize a novel, 3kb insertion mutation of Hcn2 in the Tremor and Reduced Lifespan 2 (TRLS/2J) mouse that leads to complete loss of HCN2 protein, and we show that this mutation causes many phenotypes similar to other mice lacking HCN2 expression. We then demonstrate that while TRLS/2J mice have low blood glucose levels and impaired growth, dysfunction in hormonal secretion from the pancreas, pituitary, and thyroid are unlikely to lead to this phenotype. Instead, we find that homozygous TRLS/2J mice have abnormal gastrointestinal function that is characterized by less food consumption and delayed gastrointestinal transit as compared to wildtype mice. In summary, a novel mutation in HCN2 likely leads to impaired GI motility, causing the severe growth restriction seen in mice with mutations that eliminate HCN2 expression.
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Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.
Luo W, Galvan DL, Woodard LE, Dorset D, Levy S, Wilson MH
(2017) Nucleic Acids Res 45: 8411-8422
MeSH Terms: Bacterial Proteins, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Cell Line, Tumor, DNA Transposable Elements, Endonucleases, Gene Knockout Techniques, Gene Targeting, Gene Transfer Techniques, Humans, Hypoxanthine Phosphoribosyltransferase, Mutagenesis, Insertional, Recombinant Fusion Proteins, Reproducibility of Results, Transcription Activator-Like Effector Nucleases, Transcription Activator-Like Effectors, Transposases, Zinc Fingers
Show Abstract · Added September 11, 2017
Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.
Published by Oxford University Press on behalf of Nucleic Acids Research 2017.
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18 MeSH Terms
An amyotrophic lateral sclerosis-linked mutation in GLE1 alters the cellular pool of human Gle1 functional isoforms.
Aditi , Glass L, Dawson TR, Wente SR
(2016) Adv Biol Regul 62: 25-36
MeSH Terms: Amyotrophic Lateral Sclerosis, Animals, Cytoplasm, Cytoplasmic Granules, Gene Expression, HeLa Cells, Humans, Mutagenesis, Insertional, Nuclear Envelope, Nucleocytoplasmic Transport Proteins, Phytic Acid, Point Mutation, Protein Aggregates, Protein Isoforms, RNA, Small Interfering
Show Abstract · Added February 15, 2016
Amyotrophic lateral sclerosis (ALS) is a lethal late onset motor neuron disease with underlying cellular defects in RNA metabolism. In prior studies, two deleterious heterozygous mutations in the gene encoding human (h)Gle1 were identified in ALS patients. hGle1 is an mRNA processing modulator that requires inositol hexakisphosphate (IP) binding for function. Interestingly, one hGLE1 mutation (c.1965-2A>C) results in a novel 88 amino acid C-terminal insertion, generating an altered protein. Like hGle1A, at steady state, the altered protein termed hGle1-IVS14-2A>C is absent from the nuclear envelope rim and localizes to the cytoplasm. hGle1A performs essential cytoplasmic functions in translation and stress granule regulation. Therefore, we speculated that the ALS disease pathology results from altered cellular pools of hGle1 and increased cytoplasmic hGle1 activity. GFP-hGle1-IVS14-2A>C localized to stress granules comparably to GFP-hGle1A, and rescued stress granule defects following siRNA-mediated hGle1 depletion. As described for hGle1A, overexpression of the hGle1-IVS14-2A>C protein also induced formation of larger SGs. Interestingly, hGle1A and the disease associated hGle1-IVS14-2A>C overexpression induced the formation of distinct cytoplasmic protein aggregates that appear similar to those found in neurodegenerative diseases. Strikingly, the ALS-linked hGle1-IVS14-2A>C protein also rescued mRNA export defects upon depletion of endogenous hGle1, acting in a potentially novel bi-functional manner. We conclude that the ALS-linked hGle1-c.1965-2A>C mutation generates a protein isoform capable of both hGle1A- and hGle1B-ascribed functions, and thereby uncoupled from normal mechanisms of hGle1 regulation.
Copyright © 2015 Elsevier Ltd. All rights reserved.
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15 MeSH Terms
Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity.
LaFrance ME, Farrow MA, Chandrasekaran R, Sheng J, Rubin DH, Lacy DB
(2015) Proc Natl Acad Sci U S A 112: 7073-8
MeSH Terms: Analysis of Variance, Antibodies, Monoclonal, Bacterial Proteins, Bacterial Toxins, Caco-2 Cells, Cell Adhesion Molecules, Clostridium difficile, Colon, Enterotoxins, Epithelial Cells, Genetic Complementation Test, HeLa Cells, Humans, Mutagenesis, Insertional, Nectins
Show Abstract · Added September 28, 2015
Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.
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15 MeSH Terms
The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.
Rumah KR, Ma Y, Linden JR, Oo ML, Anrather J, Schaeren-Wiemers N, Alonso MA, Fischetti VA, McClain MS, Vartanian T
(2015) PLoS Pathog 11: e1004896
MeSH Terms: Animals, Bacterial Toxins, Binding Sites, CHO Cells, Cell Death, Clostridium perfringens, Cricetulus, Humans, Injections, Intravenous, Ligands, Mice, Inbred C57BL, Mice, Knockout, Mutagenesis, Insertional, Myelin and Lymphocyte-Associated Proteolipid Proteins, Protein Interaction Domains and Motifs, Protein Precursors, Rats, Recombinant Fusion Proteins, Recombinant Proteins, Tissue Distribution, Toxicokinetics
Show Abstract · Added September 28, 2015
Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.
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21 MeSH Terms
A piggyBac insertion disrupts Foxl2 expression that mimics BPES syndrome in mice.
Shi F, Ding S, Zhao S, Han M, Zhuang Y, Xu T, Wu X
(2014) Hum Mol Genet 23: 3792-800
MeSH Terms: Animals, Blepharophimosis, DNA Transposable Elements, Disease Models, Animal, Forkhead Box Protein L2, Forkhead Transcription Factors, Humans, Maxilla, Mice, Mutagenesis, Insertional, Skin Abnormalities, Urogenital Abnormalities
Show Abstract · Added September 26, 2018
Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by small palpebral fissures and other craniofacial malformations, often with (type I) but could also without (type II) premature ovarian failure. While mutations of the forkhead transcription factor FOXL2 are associated with and likely be responsible for many BPES cases, how FOXL2 affects craniofacial development remain to be understood. Through a large-scale piggyBac (PB) insertion mutagenesis, we have identified a mouse mutant carrying a PB insertion ∼160 kb upstream of the transcription start site (TSS) of Foxl2. The insertion reduces, but not eliminates, the expression of Foxl2. This mutant, but not its revertant, displays BPES-like conditions such as midface hypoplasia, eyelid abnormalities and female subfertility. Further analysis indicates that the mutation does not affect mandible, but causes premature fusion of the premaxilla-maxilla suture, smaller premaxilla and malformed maxilla during midface development. We further identified an evolutionarily conserved fragment near the insertion site and observed enhancer activity of this element in tissue culture cells. Analyses using DNase I hypersensitivity assay and chromosome conformation capture assay in developing maxillary and periocular tissues suggest that the DNA region near the insertion site likely interacts with Foxl2 TSS. Therefore, this mutant presents an excellent animal model for mechanistic study of BPES and regulation of Foxl2.
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Targeting piggyBac transposon integrations in the human genome.
Galvan DL, Kettlun CS, Wilson MH
(2014) Methods Mol Biol 1114: 143-61
MeSH Terms: DNA Transposable Elements, Gene Order, Gene Targeting, Genetic Vectors, Genome, Human, HEK293 Cells, Homologous Recombination, Humans, Mutagenesis, Insertional
Show Abstract · Added March 6, 2014
DNA based transposon systems offer a technology for active and efficient delivery of genes into human cells. An emerging field is directed at manipulating such systems to achieve site-directed integration as compared to un-targeted integration which occurs with native or unmodified transposon systems. The naturally active piggyBac transposon system is derived from insects but has been shown to be very efficient in gene-modifying human cells. Recent efforts have utilized the fusion of DNA binding domains to the piggyBac transposase enzyme with the goal of targeting integration to specific locations in the human genome. In this chapter, we describe methodology for engineering and characterizing chimeric piggyBac transposase enzymes, including experimental approaches for evaluating activity and targeting specificity in the human genome.
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9 MeSH Terms
Conditional gene-trap mutagenesis in zebrafish.
Maddison LA, Li M, Chen W
(2014) Methods Mol Biol 1101: 393-411
MeSH Terms: Alleles, Animals, Base Sequence, DNA Transposable Elements, Embryo Culture Techniques, Female, Genetic Vectors, Male, Microinjections, Mutagenesis, Insertional, Organ Specificity, Phenotype, Zebrafish
Show Abstract · Added April 24, 2014
Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.
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13 MeSH Terms
Powerful genetic resource for the study of methicillin-resistant Staphylococcus aureus.
Hammer ND, Skaar EP
(2013) MBio 4:
MeSH Terms: Gene Knockout Techniques, Gene Library, Genetics, Microbial, Humans, Methicillin-Resistant Staphylococcus aureus, Mutagenesis, Insertional
Show Abstract · Added February 11, 2016
In "A Genetic resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes" (mBio 4(2):e00537-12, doi: 10.1128/mBio.00537-12, 2013), Fey et al. describe the creation and application of a defined transposon mutant library of methicillin-resistant S. aureus. This library is well organized and made accessible to the research community through an easily navigable central repository. The mutant library promises to be a significant resource for researchers seeking a greater understanding of this pathogen.
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6 MeSH Terms
Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin.
Zhuang T, Chen Q, Cho MK, Vishnivetskiy SA, Iverson TM, Gurevich VV, Sanders CR
(2013) Proc Natl Acad Sci U S A 110: 942-7
MeSH Terms: Arrestin, Binding Sites, Humans, Kinetics, Models, Molecular, Multiprotein Complexes, Mutagenesis, Insertional, Nuclear Magnetic Resonance, Biomolecular, Opsins, Phosphorylation, Photochemical Processes, Protein Binding, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Proteins, Rhodopsin
Show Abstract · Added December 10, 2013
Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 μM), its affinity for P-Rh (K(D) ~80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of~50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.
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16 MeSH Terms