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https://www.readbyqxmd.com/read/29071214/a-protocol-for-preparation-and-transfection-of-rat-entorhinal-cortex-organotypic-cultures-for-electrophysiological-whole-cell-recordings
#1
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
#2
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
https://www.readbyqxmd.com/read/28986907/virus-induced-gene-silencing-in-maize-with-a-foxtail-mosaic-virus-vector
#3
Yu Mei, Steven A Whitham
Virus-induced gene silencing (VIGS) is a powerful technology for rapidly and transiently knocking down the expression of plant genes to study their functions. A VIGS vector for maize derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus, was recently developed. A cloning site created near the 3' end of the FoMV genome enables insertion of 200-400 nucleotide fragments of maize genes targeted for silencing. The recombinant FoMV clones are inoculated into leaves of maize seedlings by biolistic particle delivery, and silencing is typically observed within 2 weeks after inoculation...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28986903/advances-in-agrobacterium-mediated-maize-transformation
#4
Heng Zhong, Sivamani Elumalai, Samson Nalapalli, Lee Richbourg, Anna Prairie, David Bradley, Shujie Dong, Xiujuan Jenny Su, Weining Gu, Tim Strebe, Liang Shi, Qiudeng Que
One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28986902/agrobacterium-and-biolistic-mediated-transformation-of-maize-b104-inbred
#5
Jennifer A Raji, Bronwyn Frame, Daniel Little, Tri Joko Santoso, Kan Wang
Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28974205/methods-for-genetic-transformation-of-filamentous-fungi
#6
REVIEW
Dandan Li, Yu Tang, Jun Lin, Weiwen Cai
Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation...
October 3, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28913803/doubled-haploid-transgenic-wheat-lines-by-microspore-transformation
#7
Sachin Rustgi, Nii O Ankrah, Rhoda A T Brew-Appiah, Yue Sun, Weiguo Liu, Diter von Wettstein
Microspores are preferred explant choice for genetic transformation, as their use shortens the duration of obtaining homozygous transformants. All established gene-delivery methods of particle bombardment, electroporation, and cocultivation with Agrobacterium tumefaciens were optimized on androgenic microspores or derived tissues. In the biolistic gene delivery method 35-40 days old haploid microspore embryoids were used for genetic transformation, whereas freshly isolated androgenic microspores were used for genetic transformation in the electroporation and Agrobacterium cocultivation-based methods...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28913799/biolistic-transformation-of-wheat
#8
Caroline Tassy, Pierre Barret
The wheat genome encodes some 100,000 genes. To understand how the expression of these genes is regulated it will be necessary to carry out many genetic transformation experiments. Robust protocols that allow scientists to transform a wide range of wheat genotypes are therefore required. In this chapter, we describe a protocol for biolistic transformation of wheat that uses immature embryos and small quantities of DNA cassettes. An original method for DNA cassette purification is also described. This protocol can be used to transform a wide range of wheat genotypes and other related species...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28904403/an-in-planta-biolistic-method-for-stable-wheat-transformation
#9
Haruyasu Hamada, Qianyan Linghu, Yozo Nagira, Ryuji Miki, Naoaki Taoka, Ryozo Imai
The currently favoured method for wheat (Triticum aestivum L.) transformation is inapplicable to many elite cultivars because it requires callus culture and regeneration. Here, we developed a simple, reproducible, in planta wheat transformation method using biolistic DNA delivery without callus culture or regeneration. Shoot apical meristems (SAMs) grown from dry imbibed seeds were exposed under a microscope and subjected to bombardment with different-sized gold particles coated with the GFP gene construct, introducing DNA into the L2 cell layer...
September 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28861822/transient-expression-of-chimeric-fluorescent-reporter-proteins-in-pollen-tubes-to-study-protein-polar-secretion-and-dynamics
#10
Guitao Zhong, Ronghe Liu, Menglong Zhuang, Hao Wang
Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815500/nanoparticle-mediated-recombinase-delivery-into-maize
#11
Susana Martin-Ortigosa, Brian G Trewyn, Kan Wang
We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28791544/infectivity-of-deinbollia-mosaic-virus-a-novel-weed-infecting-begomovirus-in-east-africa
#12
Martina Kyallo, Elijah Miinda Ateka, Peter Sseruwagi, José Trinidad Ascencio-Ibáñez, Mildred-Ochwo Ssemakula, Robert Skilton, Joseph Ndunguru
Weed-infecting begomoviruses play an important role in the epidemiology of crop diseases because they can potentially infect crops and contribute to the genetic diversity of crop-infecting begomoviruses. Despite the important epidemiological role that weed-infecting begomoviruses play, they remain insufficiently studied in Africa. Recently, we identified Deinbollia mosaic virus (DMV), a distinct begomovirus found naturally infecting the weed host Deinbollia borbonica (Sapindaceae) in Kenya and Tanzania. In this study, we investigated the capacity of DMV to infect a restricted host range of Solanaceae and Euphorbiaceae species...
November 2017: Archives of Virology
https://www.readbyqxmd.com/read/28667403/molecular-and-fish-analyses-of-a-53-kbp-intact-dna-fragment-inserted-by-biolistics-in-wheat-triticum-aestivum-l-genome
#13
A Partier, G Gay, C Tassy, M Beckert, C Feuillet, P Barret
A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene...
October 2017: Plant Cell Reports
https://www.readbyqxmd.com/read/28539970/developing-transgenic-wheat-to-encounter-rusts-and-powdery-mildew-by-overexpressing-barley-chi26-gene-for-fungal-resistance
#14
Hala F Eissa, Sameh E Hassanien, Ahmed M Ramadan, Moustafa M El-Shamy, Osama M Saleh, Ahmed M Shokry, Mohamed Abdelsattar, Yasser B Morsy, Maher A El-Maghraby, Hussien F Alameldin, Sabah M Hassan, Gamal H Osman, Hesham T Mahfouz, Gharib A Gad El-Karim, Magdy A Madkour, Ahmed Bahieldin
BACKGROUND: The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter was used as a selectable marker. RESULTS: Molecular analysis confirmed the integration, copy number, and the level of expression of the chi26 gene in four independent transgenic events...
2017: Plant Methods
https://www.readbyqxmd.com/read/28395198/propagation-and-some-physiological-effects-of-citrus-bark-cracking-viroid-and-apple-fruit-crinkle-viroid-in-multiple-infected-hop-humulus-lupulus-l
#15
J Matoušek, K Siglová, J Jakše, S Radišek, Joseph R J Brass, T Tsushima, T Guček, G S Duraisamy, T Sano, G Steger
The hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple viroid infections affecting hop physiology. Combinatorial biolistic hop inoculation with Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid, and Hop stunt viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses...
June 2017: Journal of Plant Physiology
https://www.readbyqxmd.com/read/28274738/labeling-of-neuronal-morphology-using-custom-diolistic-techniques
#16
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
#17
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
#18
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
#19
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
#20
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...
July 2017: Protoplasma
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