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https://www.readbyqxmd.com/read/29987734/generation-of-mutants-of-nuclear-encoded-plastid-proteins-using-crispr-cas9-in-the-diatom-phaeodactylum-tricornutum
#1
Guillaume Allorent, Erika Guglielmino, Cécile Giustini, Florence Courtois
Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29940859/a-biolistic-method-for-high-throughput-production-of-transgenic-wheat-plants-with-single-gene-insertions
#2
Ainur Ismagul, Nannan Yang, Elina Maltseva, Gulnur Iskakova, Inna Mazonka, Yuri Skiba, Huihui Bi, Serik Eliby, Satyvaldy Jatayev, Yuri Shavrukov, Nikolai Borisjuk, Peter Langridge
BACKGROUND: The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating...
June 26, 2018: BMC Plant Biology
https://www.readbyqxmd.com/read/29898980/two-distinct-approaches-for-crispr-cas9-mediated-gene-editing-in-cryptococcus-neoformans-and-related-species
#3
Ping Wang
Cryptococcus neoformans and related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility...
June 27, 2018: MSphere
https://www.readbyqxmd.com/read/29898118/genetic-transformation-of-brachiaria-brizantha-cv-marandu-by-biolistics
#4
Glaucia B Cabral, Vera T C Carneiro, Ana Cristina M M Gomes, Ana Luiza Lacerda, Adriana P Martinelli, Diva M A Dusi
Brachiaria brizantha is a forage grass well adapted to tropical areas and cultivated in millions of hectares in Brazil. The apomictic mode of reproduction in this species, in addition to differences in ploidy between sexual and apomictic plants, impairs crossbreeding. The development of a methodology to transform apomictic cultivars will provide an option to introduce agronomic important traits to B. brizantha cv. Marandu. In addition, it will open the possibility to study in vivo the function of candidate genes involved in the apomictic reproduction...
April 2018: Anais da Academia Brasileira de Ciências
https://www.readbyqxmd.com/read/29696028/engineering-the-chloroplast-genome-of-oleaginous-marine-microalga-nannochloropsis-oceanica
#5
Qinhua Gan, Jiaoyun Jiang, Xiao Han, Shifan Wang, Yandu Lu
Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation). Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established...
2018: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29516482/generation-of-high-specificity-antibodies-against-membrane-proteins-using-dna-gold-micronanoplexes-for-gene-gun-immunization
#6
Debra T Hansen, Felicia M Craciunescu, Petra Fromme, Stephen A Johnston, Kathryn F Sykes
Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA-based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA-gold micronanoplexes, which are complexes of micron-sized gold particles that allow dermal penetration and nanometer-sized gold particles that provide a higher surface area for DNA binding than micron gold alone...
February 21, 2018: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/29484813/plant-genetics-enters-the-nano-age
#7
Dirk Joldersma, Zhongchi Liu
Plant transformation has for many years relied on agrobacterium infection or biolistic particle delivery. However, these two methods are limited to model plant systems or a small number of crop species. This commentary highlights recent developments in the nanoparticle-mediated transformation that have the potential to revolutionize how plants are transformed.
June 2018: Journal of Integrative Plant Biology
https://www.readbyqxmd.com/read/29484279/construction-of-chloroplast-transformation-vector-and-its-functional-evaluation-in-momordica-charantia-l
#8
Muralikrishna Narra, Srinivas Kota, Yashodhara Velivela, Raghu Ellendula, V Rao Allini, Sadanandam Abbagani
Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn 16- rrn 23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+ II as a backbone by inserting the rrn 16- trnI and trnA - rrn 23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3'-adenylyltransferase with psbA controlling elements is added into the trnI - trnA intergenic region that confers resistance to spectinomycin...
March 2018: 3 Biotech
https://www.readbyqxmd.com/read/29458863/genetic-manipulation-of-fonsecaea-pedrosoi-using-particles-bombardment-and-agrobacterium-mediated-transformation
#9
Camille Silva Florencio, Fabiana Alves Silva Brandão, Marcus de Mello Teixeira, Anamélia Lorenzetti Bocca, Maria Sueli S Felipe, Vânia Aparecida Vicente, Larissa Fernandes
Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters...
March 2018: Microbiological Research
https://www.readbyqxmd.com/read/29444806/multiple-applications-of-a-transient-crispr-cas9-coupled-with-electroporation-trace-system-in-the-cryptococcus-neoformans-species-complex
#10
Yumeng Fan, Xiaorong Lin
Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low...
April 2018: Genetics
https://www.readbyqxmd.com/read/29408931/inhibition-of-experimental-autoimmune-encephalomyelitis-by-tolerance-promoting-dna-vaccination-focused-to-dendritic-cells
#11
Timo Castor, Nir Yogev, Thomas Blank, Christina Barwig, Marco Prinz, Ari Waisman, Matthias Bros, Angelika B Reske-Kunz
In this study we analysed the effects of prophylactic biolistic DNA vaccination with plasmids encoding the encephalitogenic protein myelin oligodendrocyte glycoprotein (MOG) on the severity of a subsequently MOGp35-55-induced EAE and on the underlying immune response. We compared the outcome of vaccination with MOG-encoding plasmids alone or in combination with vectors encoding the regulatory cytokines IL-10 and TGF-ß1, respectively. MOG expression was restricted to skin dendritic cells (DCs) by the use of the DC-specific promoter of the fascin1 gene (pFscn-MOG)...
2018: PloS One
https://www.readbyqxmd.com/read/29388938/genome-editing-of-bread-wheat-using-biolistic-delivery-of-crispr-cas9-in-vitro-transcripts-or-ribonucleoproteins
#12
Zhen Liang, Kunling Chen, Yi Zhang, Jinxing Liu, Kangquan Yin, Jin-Long Qiu, Caixia Gao
This protocol is an extension to: Nat. Protoc. 9, 2395-2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment...
March 2018: Nature Protocols
https://www.readbyqxmd.com/read/29339969/a-new-toolset-for-protein-expression-and-subcellular-localization-studies-in-citrus-and-its-application-to-citrus-tristeza-virus-proteins
#13
Amit Levy, Choaa El-Mochtar, Chunxia Wang, Michael Goodin, Vladimir Orbovic
Background: Transient gene expression is a powerful tool to study gene function in plants. In citrus, Agrobacterium transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to Agrobacterium transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than Agrobacterium ...
2018: Plant Methods
https://www.readbyqxmd.com/read/29322084/data-on-a-delivery-of-biomolecules-into-nicothiana-benthamiana-leaves-using-different-nanoparticles
#14
Antonida V Makhotenko, Ekaterina A Snigir, Natalia O Kalinina, Valentin V Makarov, Michael E Taliansky
Nanoparticles (NPs) have a number of unique properties associated with their ultrasmall size and exhibit many advantages compared with existing plant biotechnology platforms for delivery of proteins, RNA and DNA of various sizes into the plant cells (Arruda et al., 2015; Silva et al., 2010; Martin-Ortigosa et al., 2014; Mitter et al., 2017) [1], [2], [3], [4]. The data presented in this article demonstrate a delivery of biomolecules into Nicotiana benthamiana plant leaves using various types of NPs including gold, iron oxide and chitosan NPs and methods of biolistic bombardment and infiltration...
February 2018: Data in Brief
https://www.readbyqxmd.com/read/29230048/author-correction-an-in-planta-biolistic-method-for-stable-wheat-transformation
#15
Haruyasu Hamada, Qianyan Linghu, Yozo Nagira, Ryuji Miki, Naoaki Taoka, Ryozo Imai
A correction to this article has been published and is linked from the HTML version of this paper. The error has not been fixed in the paper.
December 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29209588/efficient-genetic-transformation-of-momordica-charantia-l-by-microprojectile-bombardment
#16
Muralikrishna Narra, Raghu Ellendula, Srinivas Kota, Bharathkumar Kalva, Yashodhara Velivela, Sadanandam Abbagani
Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6 µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene ( GUS ) and neomycin phosphotransferase II gene ( npt II) as a reporter and as a selectable marker gene, respectively...
January 2018: 3 Biotech
https://www.readbyqxmd.com/read/29190026/biolistic-labeling-of-retinal-ganglion-cells
#17
Luca Della Santina, Yvonne Ou
Labeling of cellular structures is of fundamental importance in the investigation of diseases of the central nervous system. Biolistic labeling of retinal ganglion cells (RGCs) allows visualization of dendritic and synaptic structures of RGCs in retinal explants from animal models of experimental glaucoma. This technique sparsely labels RGCs, and, due to the stochastic nature of the particle delivery, all RGC types can be potentially observed in the labeled tissue. Quantification of dendritic and synaptic properties permits examination of the specific alterations to RGC morphology at different stages of degeneration, such as dendritic shrinkage and excitatory synapse loss...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29170630/heterogeneity-of-the-axon-initial-segment-in-interneurons-and-pyramidal-cells-of-rodent-visual-cortex
#18
Felix Höfflin, Alexander Jack, Christian Riedel, Julia Mack-Bucher, Johannes Roos, Corinna Corcelli, Christian Schultz, Petra Wahle, Maren Engelhardt
The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/29071214/a-protocol-for-preparation-and-transfection-of-rat-entorhinal-cortex-organotypic-cultures-for-electrophysiological-whole-cell-recordings
#19
Nicholas I Cilz, James E Porter, Saobo Lei
Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC) is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices...
2017: MethodsX
https://www.readbyqxmd.com/read/29039016/application-of-tissue-culture-and-transformation-techniques-in-model-species-brachypodium-distachyon
#20
Bahar Sogutmaz Ozdemir, Hikmet Budak
Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation...
2018: Methods in Molecular Biology
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