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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
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 development in the nanoparticle-mediated transformation that has the potential to revolutionize how plants are transformed.
February 27, 2018: Journal of Integrative Plant Biology
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
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
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 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...
February 14, 2018: Genetics
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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