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Bio-protocol

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https://www.readbyqxmd.com/read/28804739/selection-of-genetically-modified-bacteriophages-using-the-crispr-cas-system
#1
Miriam Manor, Udi Qimron
We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28781989/isolation-of-ustilago-bromivora-strains-from-infected-spikelets-through-spore-recovery-and-germination
#2
Jason Bosch, Armin Djamei
Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly use this spore material to infect Brachypodium seeds, in many cases it is more useful to isolate individual strains of U. bromivora for a genetically homogenous population...
July 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28804738/social-observation-task-in-a-linear-maze-for-rats
#3
Xiang Mou, Daoyun Ji
Animals often learn through observing their conspecifics. However, the mechanisms of them obtaining useful knowledge during observation are beginning to be understood. This protocol describes a novel social observation task to test the 'local enhancement theory', which proposes that presence of social subjects in an environment facilitates one's understanding of the environments. By combining behavior test and in vivo electrophysiological recording, we found that social observation can facilitate the observer's spatial representation of an unexplored environment...
July 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28798946/ex-vivo-ooplasmic-extract-from-developing-drosophila-oocytes-for-quantitative-tirf-microscopy-analysis
#4
Imre Gaspar, Anne Ephrussi
Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision...
July 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28748203/thermal-stability-of-heterotrimeric-pmhc-proteins-as-determined-by-circular-dichroism-spectroscopy
#5
Anna Fuller, Aaron Wall, Michael D Crowther, Angharad Lloyd, Alexei Zhurov, Andrew K Sewell, David K Cole, Konrad Beck
T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease...
July 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28798945/optogenetic-stimulation-and-recording-of-primary-cultured-neurons-with-spatiotemporal-control
#6
Jérémie Barral, Alex D Reyes
We studied a network of cortical neurons in culture and developed an innovative optical device to stimulate optogenetically a large neuronal population with both spatial and temporal precision. We first describe how to culture primary neurons expressing channelrhodopsin. We then detail the optogenetic setup based on the workings of a fast Digital Light Processing (DLP) projector. The setup is able to stimulate tens to hundreds neurons with independent trains of light pulses that evoked action potentials with high temporal resolution...
June 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28758141/isolation-and-infection-of-drosophila-primary-hemocytes
#7
Charles Tracy, Helmut Krämer
Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and degradation within individual hemocytes over time.
June 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28748202/the-repeated-flurothyl-seizure-model-in-mice
#8
Russell J Ferland
Development of spontaneous seizures is the hallmark of human epilepsy. There is a critical need for new epilepsy models in order to elucidate mechanisms responsible for leading to the development of spontaneous seizures and for testing new anti-epileptic compounds. Moreover, rodent models of epilepsy have clearly demonstrated that there are two independent seizure systems in the brain: 1) the forebrain seizure network required for the expression of clonic seizures mediated by forebrain neurocircuitry, and 2) the brainstem seizure network necessary for the expression of brainstem or tonic seizures mediated by brainstem neurocircuitry...
June 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28758140/endogenous-c-terminal-tagging-by-crispr-cas9-in-trypanosoma-cruzi
#9
Noelia Lander, Miguel A Chiurillo, Aníbal E Vercesi, Roberto Docampo
To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3' end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3' end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus...
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28758139/assaying-the-effects-of-splice-site-variants-by-exon-trapping-in-a-mammalian-cell-line
#10
Stuart W Tompson, Terri L Young
There are several in silico programs that endeavor to predict the functional impact of an individual's sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products...
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28752107/muscle-histology-characterization-using-h-e-staining-and-muscle-fiber-type-classification-using-immunofluorescence-staining
#11
Chao Wang, Feng Yue, Shihuan Kuang
Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber diameter, the presence of degenerated and regenerated myofibers, and adipocytes and fibrotic cells. Muscle fibers in adult skeletal muscles of rodents are classified into 4 subtypes based on the expression of myosin heavy chain proteins: Myh7 (type I fiber), Myh2 (type IIA fiber), Myh1 (type IIX fiber), Myh4 (type IIB fiber)...
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28603749/isolation-and-cultivation-of-primary-brain-endothelial-cells-from-adult-mice
#12
Julian Christopher Assmann, Kristin Müller, Jan Wenzel, Thomas Walther, Josefine Brands, Peter Thornton, Stuart M Allan, Markus Schwaninger
Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected by puromycin treatment and grown to confluence within one week.
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28573168/analysis-of-in-vivo-interaction-between-rna-binding-proteins-and-their-rna-targets-by-uv-cross-linking-and-immunoprecipitation-clip-method
#13
Pamela Bielli, Claudio Sette
RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant...
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28752111/olfactory-cued-learning-paradigm
#14
Gary Liu, Cynthia K McClard, Burak Tepe, Jessica Swanson, Brandon Pekarek, Sugi Panneerselvam, Benjamin R Arenkiel
Sensory stimulation leads to structural changes within the CNS (Central Nervous System), thus providing the fundamental mechanism for learning and memory. The olfactory circuit offers a unique model for studying experience-dependent plasticity, partly due to a continuous supply of integrating adult born neurons. Our lab has recently implemented an olfactory cued learning paradigm in which specific odor pairs are coupled to either a reward or punishment to study downstream circuit changes. The following protocol outlines the basic set up for our learning paradigm...
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28752110/escherichia-coli-infection-of-drosophila
#15
Charles Tracy, Helmut Krämer
Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection...
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28752109/conjugation-assay-for-testing-crispr-cas-anti-plasmid-immunity-in-staphylococci
#16
Forrest C Walker, Asma Hatoum-Aslan
CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities...
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28730161/isolation-culturing-and-differentiation-of-primary-myoblasts-from-skeletal-muscle-of-adult-mice
#17
Lubna Hindi, Joseph D McMillan, Dil Afroze, Sajedah M Hindi, Ashok Kumar
Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes...
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28725659/explant-methodology-for-analyzing-neuroblast-migration
#18
Kirsty J Dixon, Alisa Turbic, Ann M Turnley, Daniel J Liebl
The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28660237/analysis-of-replicative-intermediates-of-adeno-associated-virus-through-hirt-extraction-and-southern-blotting
#19
Martino Bardelli, Francisco Zarate-Perez, Leticia Agundez, Nelly Jolinon, R Michael Linden, Carlos R Escalante, Els Henckaerts
Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975)...
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28534038/analysis-of-mitochondrial-transfer-in-direct-co-cultures-of-human-monocyte-derived-macrophages-mdm-and-mesenchymal-stem-cells-msc
#20
Megan V Jackson, Anna D Krasnodembskaya
Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence...
May 5, 2017: Bio-protocol
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