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Electroporation

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https://www.readbyqxmd.com/read/28640817/an-r-camp1-07-reporter-mouse-for-cell-type-specific-expression-of-a-sensitive-red-fluorescent-calcium-indicator
#1
Philipp Bethge, Stefano Carta, Dayra A Lorenzo, Ladan Egolf, Despoina Goniotaki, Linda Madisen, Fabian F Voigt, Jerry L Chen, Bernard Schneider, Masamichi Ohkura, Junichi Nakai, Hongkui Zeng, Adriano Aguzzi, Fritjof Helmchen
Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1...
2017: PloS One
https://www.readbyqxmd.com/read/28636433/immunogenicity-of-ags-004-dendritic-cell-therapy-in-patients-treated-during-acute-hiv-infection
#2
Cynthia L Gay, Mark A DeBenedette, Irina Y Tcherepanova, Alicia Gamble, Whitney E Lewis, Anna B Cope, JoAnn D Kuruc, Kara S McGee, Mary F Kearney, John M Coffin, Nancie M Archin, Charles B Hicks, Joseph J Eron, Charles A Nicolette, David M Margolis
AGS-004 consists of matured autologous dendritic cells co-electroporated with in vitro transcribed RNA encoding autologous HIV antigens. In an open-label, single arm sub-study of AGS-004-003, AGS-004 was administered monthly to suppressed participants who started antiretroviral therapy (ART) during acute HIV infection. HIV-1 specific T cell responses were measured by multicolor flow cytometry after 3-4 doses. The frequency of resting CD4(+) T-cell infection (RCI) was measured by quantitative viral outgrowth assay...
June 21, 2017: AIDS Research and Human Retroviruses
https://www.readbyqxmd.com/read/28633406/in-saccharomyces-cerevisiae-gene-targeting-fidelity-depends-on-a-transformation-method-and-proportion-of-the-overall-length-of-the-transforming-and-targeted-dna
#3
Anamarija Štafa, Marina Svetec Miklenic, Antonio Zandona, Bojan Žunar, Neža Cadež, Hrvoje Petkovic, Ivan Krešimir Svetec
Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome...
June 15, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28631386/toward-establishing-model-organisms-for-marine-protists-successful-transfection-protocols-for-parabodo-caudatus-kinetoplastida-excavata
#4
Fatma Gomaa, Paulo A Garcia, Jennifer Delaney, Peter R Girguis, Cullen R Buie, Virginia P Edgcomb
We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: 1) a square-wave electroporator designed for eukaryotes, 2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and 3) a traditional exponential decay electroporator...
June 19, 2017: Environmental Microbiology
https://www.readbyqxmd.com/read/28630163/measuring-the-potential-energy-barrier-to-lipid-bilayer-electroporation
#5
Jason T Sengel, Mark I Wallace
Electroporation is a common tool for gene transfection, tumour ablation, sterilization and drug delivery. Using experimental methods, we explore the temperature dependence of electropore formation in a model membrane system (droplet-interface bilayers), using optical single-channel recording to image the real-time gating of individual electropores. We investigate the influence of the agarose substrate on electropores formed in this system. Furthermore, by examining the temperature-dependent kinetics of pore opening and closure we are able to estimate a barrier to pore opening in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes to be 25...
August 5, 2017: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
https://www.readbyqxmd.com/read/28626144/glass-bead-based-genetic-transformation-an-efficient-method-for-transformation-of-thraustochytrid-microorganisms
#6
Takumi Adachi, Takehiko Sahara, Hidetoshi Okuyama, Naoki Morita
Here, we describe a new method for genetic transformation of thraustochytrids, well-known producers of polyunsaturated fatty acids (PUFAs) like docosahexaenoic acid, by combining mild glass (zirconia) bead treatment and electroporation. Because the cell wall is a barrier against transfer of exogenous DNA into cells, gentle vortexing of cells with glass beads was performed prior to electroporation for partial cell wall disruption. G418-resistant transformants of thraustochytrid cells (Aurantiochytrium limacinum strain SR21 and thraustochytrid strain 12B) were successfully obtained with good reproducibility...
June 13, 2017: Journal of Oleo Science
https://www.readbyqxmd.com/read/28624558/safety-and-feasibility-of-electrochemotherapy-in-patients-with-unresectable-colorectal-liver-metastases-a-pilot-study
#7
Laura Coletti, Valentina Battaglia, Paolo De Simone, Laura Turturici, Carlo Bartolozzi, Franco Filipponi
BACKGROUND AND OBJECTIVES: Electrochemotherapy is a novel ablation technique combining chemotherapeutic agents with reversible cell membrane electroporation. Previous experiences have shown its efficacy for cutaneous tumors. Its application for deep-seated malignancies is under investigation. We performed a prospective, pilot study to evaluate the feasibility, safety, and efficacy of intraoperative electrochemotherapy for otherwise unresectable colorectal liver metastases. METHODS: Electrochemotherapy with bleomycin was combined with open liver resection and performed with linear or hexagonal needle electrodes according to an individualized pretreatment plan...
June 14, 2017: International Journal of Surgery
https://www.readbyqxmd.com/read/28621430/disruption-of-the-aortic-wall-by-coelomic-lining-derived-mesenchymal-cells-accompanies-the-onset-of-aortic-hematopoiesis
#8
Alaa A Arraf, Marella F T R De Bruijn, Thomas M Schultheiss
In vertebrates, definitive hematopoietic stem cells (HSCs) first emerge in the ventral wall of the aorta in the Aorta-Gonad-Mesonephros (AGM) region of the embryo, where they differentiate from a specialized type of endothelium termed Hemogenic Endothelium (HE). While the transition from HE to hematopoietic tissue has received much experimental attention, much less is known regarding generation of HE itself. The current study investigates the emergence of the HE in the chick embryo aorta. Using the HE marker Runx1 as well as a new chicken-reactive antibody to the endothelial marker VE-Cadherin, we document the relationship between the emerging HE and surrounding tissues, particularly the coelomic epithelium (CE) and CE-derived sub-aortic mesenchyme...
2017: International Journal of Developmental Biology
https://www.readbyqxmd.com/read/28620838/modelling-fus-mislocalisation-in-an-in-vitro-model-of-innervated-human-muscle
#9
Sonja Prpar Mihevc, Mojca Pavlin, Simona Darovic, Marko Živin, Matej Podbregar, Boris Rogelj, Tomaz Mars
Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS...
June 15, 2017: Journal of Molecular Neuroscience: MN
https://www.readbyqxmd.com/read/28615204/human-cytomegalovirus-ie2-protein-disturbs-brain-development-by-dysregulating-neural-stem-cell-maintenance-and-the-polarization-of-migrating-neurons
#10
Dasol Han, Sung-Hyun Byun, Juwan Kim, Mookwang Kwon, Samuel J Pleasure, Jin-Hyun Ahn, Keejung Yoon
Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2...
June 14, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28604083/implantation-of-induced-pluripotent-stem-cell-derived-tracheal-epithelial-cells
#11
Masakazu Ikeda, Mitsuyoshi Imaizumi, Susumu Yoshie, Ryosuke Nakamura, Koshi Otsuki, Shigeyuki Murono, Koichi Omori
OBJECTIVES: Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects...
July 2017: Annals of Otology, Rhinology, and Laryngology
https://www.readbyqxmd.com/read/28602961/progress-and-biotechnological-prospects-in-fish-transgenesis
#12
REVIEW
Fernanda M P Tonelli, Samyra M S N Lacerda, Flávia C P Tonelli, Guilherme M J Costa, Luiz Renato de França, Rodrigo R Resende
The history of transgenesis is marked by milestones such as the development of cellular transdifferentiation, recombinant DNA, genetic modification of target cells, and finally, the generation of simpler genetically modified organisms (e.g. bacteria and mice). The first transgenic fish was developed in 1984, and since then, continuing technological advancements to improve gene transfer have led to more rapid, accurate, and efficient generation of transgenic animals. Among the established methods are microinjection, electroporation, lipofection, viral vectors, and gene targeting...
June 8, 2017: Biotechnology Advances
https://www.readbyqxmd.com/read/28599791/characterization-of-hpv18-e6-specific-t-cell-responses-and-establishment-of-hpv18-e6-expressing-tumor-model
#13
Ying Ma, Andrew Yang, Shiwen Peng, Jin Qiu, Emily Farmer, Chien-Fu Hung, T-C Wu
Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer, and subsets of anogenital and oropharyngeal cancers. HPV18 is the second most prevalent high-risk HPV type after HPV16. Furthermore, HPV18 is responsible for approximately 12% of cervical squamous cell carcinoma and 37% of cervical adenocarcinoma cases worldwide. In this study, we aimed to characterize the HPV18-E6-specific epitope and establish an HPV18 animal tumor model to evaluate the E6-specific immune response induced by our DNA vaccine...
June 6, 2017: Vaccine
https://www.readbyqxmd.com/read/28598105/-genetic-recombiniation-and-protein-expression-detection-of-listeria-based-tuberculosis-vaccine-candidates
#14
Chen-Yan Ren, Si-Jing Liu, Qi-Kang Pu, Ming-Juan Jiang, Meng-Ying Zhou, Chuan Wang
OBJECTIVES: Genetic construction of tuberculosis vaccine candidates based on Listeria(L.) monocytogenes,L.ivanovii,and evaluation their protein expression,in order to provide a novel method for research on tuberculosis controlling. METHODS: Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L.monocytogenes,L.ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro...
November 2016: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition
https://www.readbyqxmd.com/read/28596588/optimization-of-a-crispr-cas9-mediated-knock-in-strategy-at-the-porcine-rosa26-locus-in-porcine-foetal-fibroblasts
#15
Zicong Xie, Daxin Pang, Kankan Wang, Mengjing Li, Nannan Guo, Hongming Yuan, Jianing Li, Xiaodong Zou, Huping Jiao, Hongsheng Ouyang, Zhanjun Li, Xiaochun Tang
Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29...
June 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28594449/predictive-therapeutic-planning-for-irreversible-electroporation-treatment-of-spontaneous-malignant-glioma
#16
Paulo A Garcia, Bor Kos, John H Rossmeisl, Denis Pavliha, Damijan Miklavčič, Rafael V Davalos
PURPOSE: Irreversible electroporation (IRE) have been developed as a promising minimally invasive treatment to ablate spontaneous brain tumors with pulsed electric fields in canine patients. The purpose of the study is to determine the Peleg-Fermi parameters needed to incorporate pulse number and pulse duration into the therapeutic planning of IRE. METHODS: Seven canine patients were treated with IRE for spontaneous malignant glioma with MRI based treatment planning...
June 8, 2017: Medical Physics
https://www.readbyqxmd.com/read/28591194/regulation-of-neural-stem-cell-proliferation-and-differentiation-by-kinesin-family-member-2a
#17
Dong Sun, Xue Zhou, Hua-Li Yu, Xiao-Xiao He, Wei-Xiang Guo, Wen-Cheng Xiong, Xiao-Juan Zhu
In the developing neocortex, cells in the ventricular/subventricular zone are largely multipotent neural stem cells and neural progenitor cells. These cells undergo self-renewal at the early stage of embryonic development to amplify the progenitor pool and subsequently differentiate into neurons. It is thus of considerable interest to investigate mechanisms controlling the switch from neural stem cells or neural progenitor cells to neurons. Here, we present evidence that Kif2a, a member of the Kinesin-13 family, plays a role in regulating the proliferation and differentiation of neural stem cells or neural progenitor cells at embryonic day 13...
2017: PloS One
https://www.readbyqxmd.com/read/28588454/reelin-signaling-inactivates-cofilin-to-stabilize-the-cytoskeleton-of-migrating-cortical-neurons
#18
REVIEW
Michael Frotscher, Shanting Zhao, Shaobo Wang, Xuejun Chai
Neurons are highly polarized cells. They give rise to several dendrites but only one axon. In addition, many neurons show a preferred orientation. For example, pyramidal neurons of the cerebral cortex extend their apical dendrites toward the cortical surface while their axons run in opposite direction toward the white matter. This characteristic orientation reflects the migratory trajectory of a pyramidal cell during cortical development: the leading process (the future apical dendrite) extends toward the marginal zone (MZ) and the trailing process (the future axon) toward the intermediate zone (IZ) while the cells migrate radially to reach their destination in the cortical plate (CP)...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28586250/pain-after-percutaneous-irreversible-electroporation-of-renal-tumors-is-not-dependent-on-tumor-location
#19
Igor Sorokin, Aaron H Lay, Nikitha K Reddy, Noah E Canvasser, Murthy Chamarthy, Jeffrey A Cadeddu
Introduction Irreversible electroporation (IRE) is a non-thermal minimally invasive technique to treat small renal masses (SRM). Prior work has demonstrated greater narcotic requirements after radiofrequency ablation (RFA) for tumors closer to body-wall musculature. We hypothesized that pain after IRE is not dependent on tumor location due to the athermal mechanistic action. Methods Retrospective review of 50 consecutive percutaneous IRE and RFA cases was performed from 2013 to 2014. Eight patients were excluded from analysis due to incomplete anesthesia record and/or multiple ablations per session leaving 21 patients in each group...
June 6, 2017: Journal of Endourology
https://www.readbyqxmd.com/read/28585227/developing-guinea-pig-brain-as-a-model-for-cortical-folding
#20
Jun Hatakeyama, Haruka Sato, Kenji Shimamura
The cerebral cortex in mammals, the neocortex specifically, is highly diverse among species with respect to its size and morphology, likely reflecting the immense adaptiveness of this lineage. In particular, the pattern and number of convoluted ridges and fissures, called gyri and sulci, respectively, on the surface of the cortex are variable among species and even individuals. However, little is known about the mechanism of cortical folding, although there have been several hypotheses proposed. Recent studies on embryonic neurogenesis revealed the differences in cortical progenitors as a critical factor of the process of gyrification...
June 5, 2017: Development, Growth & Differentiation
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