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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
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
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...
October 28, 2016: Plant Molecular Biology
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
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
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
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
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
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
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
Lílian S T Carmo, André M Murad, Renato O Resende, Leonardo S Boiteux, Simone G Ribeiro, Jesús V Jorrín-Novo, Angela Mehta
: Tomato chlorotic mottle virus (ToCMoV) is a widespread bipartite Begomovirus species found in tomato fields in Brazil. In this study, plant responses and putative mechanisms associated with the 'Tyking'-derived recessive resistance to ToCMoV were investigated. Changes in the protein profile in the inoculated plants of two near isogenic tomato lines resistant ('LAM 157') and susceptible ('Santa Clara') to ToCMoV were analyzed. Seedlings were biolistically inoculated with an infectious ToCMoV clone...
July 22, 2016: Journal of Proteomics
Weina Hou, Preeti Shakya, Gregory Franklin
Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus...
2016: Frontiers in Plant Science
John C O'Donnell, Joshua G Jackson, Michael B Robinson
UNLABELLED: Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B...
July 6, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
K M McGregor, C Bécamel, P Marin, R Andrade
Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle...
September 1, 2016: Journal of Neurophysiology
Hao Wu, Yosvanis Acanda, Hongge Jia, Nian Wang, Janice Zale
The development of transgenic citrus plants by the biolistic method. A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18...
September 2016: Plant Cell Reports
Inna A Belyantseva
The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo...
2016: Methods in Molecular Biology
Kota Srinivas, Narra Muralikrishna, Kalva Bharath Kumar, Ellendula Raghu, Aileni Mahender, Kasula Kiranmayee, Velivela Yashodahara, Abbagani Sadanandam
Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated...
January 2016: Physiology and Molecular Biology of Plants: An International Journal of Functional Plant Biology
Luana R M M Aps, Milene B Tavares, Julio H K Rozenfeld, M Teresa Lamy, Luís C S Ferreira, Mariana O Diniz
Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles...
June 20, 2016: Journal of Biotechnology
Laura Lossi, Carolina Cocito, Silvia Alasia, Adalberto Merighi
BACKGROUND: Apoptosis takes place in naturally occurring neuronal death, but also in aging, neurodegenerative disorders, and traumatic brain injuries. Caspase 3 (Casp3) is the most important effector protease in apoptosis: being inactive inside the cell, it undergoes enzymatic cleavage and - hence - activation once the apoptotic cascade is triggered. Immunological techniques with antibodies against cleaved Casp3 (cCasp3) or assays with colorimetric/fluorogenic substrates are commonly in use, but they do not allow to directly follow the dynamics of activation in alive neurons that may be committed to die...
2016: Molecular Neurodegeneration
Brad C Stone, Arnold Kas, Zachary P Billman, Deborah H Fuller, James T Fuller, Jay Shendure, Sean C Murphy
Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines...
2016: PloS One
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