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https://www.readbyqxmd.com/read/28274738/labeling-of-neuronal-morphology-using-custom-diolistic-techniques
#1
Lyon H Hough, Michael E Brown
BACKGROUND: Diolistic labeling is increasingly utilized in neuroscience as an efficient, reproducible method for visualization of neuronal morphology. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery allows for non-toxic fluorescent labeling of multiple neurons in both living and fixed tissue. Since first described, this labeling method has been modified to fit a variety of research goals and laboratory settings. NEW METHOD: Diolistic labeling has traditionally relied on commercially available devices for the propulsion of coated micro-particles into tissue sections...
March 6, 2017: Journal of Neuroscience Methods
https://www.readbyqxmd.com/read/28270156/a-rapid-virus-induced-gene-silencing-vigs-method-for-assessing-resistance-and-susceptibility-to-cassava-mosaic-disease
#2
Getu Beyene, Raj Deepika Chauhan, Nigel J Taylor
BACKGROUND: Cassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12-18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones...
March 7, 2017: Virology Journal
https://www.readbyqxmd.com/read/28148855/viral-vector-based-transduction-of-slice-cultures
#3
J Simon Wiegert, Christine E Gee, Thomas G Oertner
Transgenes can be introduced into the cells of organotypic slice cultures using different delivery methods, such as biolistic transfection, electroporation, and viral vector-based transduction. These methods produce different patterns of transgene expression. Local injection of recombinant adeno-associated virus (rAAV) produces a small cluster of transgene-expressing neurons around the injection site. Expression in individual cells varies with the distance from the injection site, indicating that many neurons take up several rAAV particles...
February 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28132145/agrobacterium-mediated-transformation-of-leaf-base-segments
#4
Sebastian Gasparis
Agrobacterium-mediated transformation has become a routine method of genetic engineering of cereals, gradually replacing the biolistic protocols. Simple integration patterns of transgenic loci, decent transformation efficiency, and technical simplicity are the main advantages offered by this method. Here we present a detailed protocol for the production of transgenic oat plants by Agrobacterium-mediated transformation of leaf base segments. The use of leaf explants as target tissues for transformation and in vitro regeneration of transgenic plants may be a good alternative for genotypes which are not susceptible to regeneration from immature or mature embryos...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28120101/virus-induced-gene-silencing-in-rauwolfia-species
#5
Cyrielle Corbin, Florent Lafontaine, Liuda Johana Sepúlveda, Ines Carqueijeiro, Martine Courtois, Arnaud Lanoue, Thomas Dugé de Bernonville, Sébastien Besseau, Gaëlle Glévarec, Nicolas Papon, Lucia Atehortúa, Nathalie Giglioli-Guivarc'h, Marc Clastre, Benoit St-Pierre, Audrey Oudin, Vincent Courdavault
Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure...
January 24, 2017: Protoplasma
https://www.readbyqxmd.com/read/28081289/transgenic-cotton-expressing-cry10aa-toxin-confers-high-resistance-to-the-cotton-boll-weevil
#6
Thuanne Pires Ribeiro, Fabricio Barbosa Monteiro Arraes, Isabela Tristan Lourenço-Tessutti, Marilia Santos Silva, Maria Eugênia Lisei-de-Sá, Wagner Alexandre Lucena, Leonardo Lima Pepino Macedo, Janaina Nascimento Lima, Regina Maria Santos Amorim, Sinara Artico, Márcio Alves-Ferreira, Maria Cristina Mattar Silva, Maria Fatima Grossi-de-Sa
Genetically modified (GM) cotton events that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation...
January 12, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28071976/precise-flexible-and-affordable-gene-stacking-for-crop-improvement
#7
Weiqiang Chen, David W Ow
Addendum to: Hou L, Yau YY, Wei J, Han Z, Dong Z, Ow DW. An open-source system for in planta gene stacking by Bxb1 and Cre recombinases. Mol Plant, 2014; 7(12):1756-1765. doi:10.1093/mp/ssu107; Li R, Han Z, Hou L, Kaur G, Yin Q, Ow DW. Method for Biolistic Site-Specific Integration in Plants Catalyzed by Bxb1 Integrase. In: Murata M, editor. Chromosome and Genomic Engineering in Plants: Methods and Protocols, Methods in Molecular Biology. 2016; p.15-30; and Chen W, Ow DW. Protocol for In Vitro Stacked Molecules Compatible with In Vivo Recombinase-Mediated Gene Stacking...
January 10, 2017: Bioengineered
https://www.readbyqxmd.com/read/28005227/in-planta-production-and-characterization-of-a-hyperthermostable-gh10-xylanase-in-transgenic-sugarcane
#8
Jae Yoon Kim, Guang Nong, John D Rice, Maria Gallo, James F Preston, Fredy Altpeter
Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes...
December 22, 2016: Plant Molecular Biology
https://www.readbyqxmd.com/read/27973750/controlled-release-and-intracellular-protein-delivery-from-mesoporous-silica-nanoparticles
#9
REVIEW
Gauri V Deodhar, Marisa L Adams, Brian G Trewyn
Protein therapeutics are promising candidates for disease treatment due to their high specificity and minimal adverse side effects; however, targeted protein delivery to specific sites has proven challenging. Mesoporous silica nanoparticles (MSN) have demonstrated to be ideal candidates for this application, given their high loading capacity, biocompatibility, and ability to protect host molecules from degradation. These materials exhibit tunable pore sizes, shapes and volumes, and surfaces which can be easily functionalized...
January 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/27943461/high-efficiency-gene-targeting-in-hexaploid-wheat-using-dna-replicons-and-crispr-cas9
#10
Javier Gil-Humanes, Yanpeng Wang, Zhen Liang, Qiwei Shan, Carmen V Ozuna, Susana Sánchez-León, Nicholas J Baltes, Colby Starker, Francisco Barro, Caixia Gao, Daniel F Voytas
The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons...
December 10, 2016: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/27848933/genome-editing-in-maize-directed-by-crispr-cas9-ribonucleoprotein-complexes
#11
Sergei Svitashev, Christine Schwartz, Brian Lenderts, Joshua K Young, A Mark Cigan
Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles...
November 16, 2016: Nature Communications
https://www.readbyqxmd.com/read/27826796/bioengineering-of-the-plant-culture-of-capsicum-frutescens-with-vanillin-synthase-gene-for-the-production-of-vanillin
#12
Marcus Jenn Yang Chee, Grantley W Lycett, Teng-Jin Khoo, Chiew Foan Chin
Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively...
November 8, 2016: Molecular Biotechnology
https://www.readbyqxmd.com/read/27796719/transformation-of-the-cyanidioschyzon-merolae-chloroplast-genome-prospects-for-understanding-chloroplast-function-in-extreme-environments
#13
Maksymilian Zienkiewicz, Tomasz Krupnik, Anna Drożak, Anna Golke, Elżbieta Romanowska
We have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism. Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae-an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene...
January 2017: Plant Molecular Biology
https://www.readbyqxmd.com/read/27750454/the-putative-autophagy-regulator-atg7-affects-the-physiology-and-pathogenic-mechanisms-of-cryptococcus-neoformans
#14
Debora L Oliveira, Fernanda L Fonseca, Daniel Zamith-Miranda, Leonardo Nimrichter, Jéssica Rodrigues, Marcos D Pereira, Julia Cv Reuwsaat, Augusto Schrank, Charley Staats, Livia Kmetzsch, Marilene H Vainstein, Marcio L Rodrigues
AIM: We investigated the involvement of the autophagy protein 7 (Atg7) in physiology and pathogenic potential of Cryptococcus neoformans. MATERIALS & METHODS: The C. neoformans gene encoding Atg7 was deleted by biolistic transformation for characterization of autophagy mechanisms, pigment formation, cell dimensions, interaction with phagocytes and pathogenic potential in vivo. RESULTS & CONCLUSION: ATG7 deletion resulted in defective autophagy mechanisms, enhanced pigmentation and increased cellular size both in vitro and in vivo...
October 18, 2016: Future Microbiology
https://www.readbyqxmd.com/read/27643854/chemical-inhibitors-of-non-homologous-end-joining-increase-targeted-construct-integration-in-cryptococcus-neoformans
#15
Samantha D M Arras, James A Fraser
The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ)...
2016: PloS One
https://www.readbyqxmd.com/read/27625678/induction-of-silencing-in-plants-by-high-pressure-spraying-of-in-vitro-synthesized-small-rnas
#16
Athanasios Dalakouras, Michèle Wassenegger, John N McMillan, Vinitha Cardoza, Ira Maegele, Elena Dadami, Miriam Runne, Gabi Krczal, Michael Wassenegger
In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction...
2016: Frontiers in Plant Science
https://www.readbyqxmd.com/read/27600536/morphogenic-regulators-baby-boom-and-wuschel-improve-monocot-transformation
#17
Keith Lowe, Emily Wu, Ning Wang, George Hoerster, Craig Hastings, Myeong-Je Cho, Chris Scelonge, Brian Lenderts, Mark Chamberlin, Josh Cushatt, Lijuan Wang, Larisa Ryan, Tanveer Khan, Julia Chow-Yiu, Wei Hua, Maryanne Yu, Jenny Banh, Zhongmeng Bao, Kent Brink, Elizabeth Igo, Bhojaraja Rudrappa, P M Shamseer, Wes Bruce, Lisa Newman, Bo Shen, Peizhong Zheng, Dennis Bidney, S Carl Falco, James C RegisterIII, Zuo-Yu Zhao, Deping Xu, Todd J Jones, William James Gordon-Kamm
While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously non-transformable maize inbred lines...
September 6, 2016: Plant Cell
https://www.readbyqxmd.com/read/27581463/selective-vulnerability-of-specific-retinal-ganglion-cell-types-and-synapses-after-transient-ocular-hypertension
#18
Yvonne Ou, Rebecca E Jo, Erik M Ullian, Rachel O L Wong, Luca Della Santina
UNLABELLED: Key issues concerning ganglion cell type-specific loss and synaptic changes in animal models of experimental glaucoma remain highly debated. Importantly, changes in the structure and function of various RGC types that occur early, within 14 d after acute, transient intraocular pressure elevation, have not been previously assessed. Using biolistic transfection of individual RGCs and multielectrode array recordings to measure light responses in mice, we examined the effects of laser-induced ocular hypertension on the structure and function of a subset of RGCs...
August 31, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/27557683/method-for-biolistic-site-specific-integration-in-plants-catalyzed-by-bxb1-integrase
#19
Ruyu Li, Zhiguo Han, Lili Hou, Gurminder Kaur, Qian Yin, David W Ow
Crop improvement is a never ending process. With a transgenesis approach, it is not inconceivable to envision a continuous addition of new transgenes to existing cultivars. Previously, we described a recombinase-directed gene stacking method in tobacco (Hou et al., Mol Plant 7:1756-1765, 2014). Being able to stack DNA to a previous location ensures that the number of genetic loci does not increase with each new round of transgene addition. Whereas the previous demonstration was conducted through polyethylene glycol to mediate uptake of DNA into tobacco protoplasts, we now describe protocols for using biolistic transformation to stack DNA in tobacco and rice...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27480687/prequels-to-synthetic-biology-from-candidate-gene-identification-and-validation-to-enzyme-subcellular-localization-in-plant-and-yeast-cells
#20
E Foureau, I Carqueijeiro, T Dugé de Bernonville, C Melin, F Lafontaine, S Besseau, A Lanoue, N Papon, A Oudin, G Glévarec, M Clastre, B St-Pierre, N Giglioli-Guivarc'h, V Courdavault
Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches...
2016: Methods in Enzymology
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