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Shashank Gandhi, Florian Razy-Krajka, Lionel Christiaen, Alberto Stolfi
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a revolutionary tool for fast and efficient targeted gene knockouts and genome editing in almost any organism. The laboratory model tunicate Ciona is no exception. Here, we describe our latest protocol for the design, implementation, and evaluation of successful CRISPR/Cas9-mediated gene knockouts in somatic cells of electroporated Ciona embryos. Using commercially available reagents, publicly accessible plasmids, and free web-based software applications, any Ciona researcher can easily knock out any gene of interest in their favorite embryonic cell lineage...
2018: Advances in Experimental Medicine and Biology
Julie E Maguire, Aakarsha Pandey, Yushi Wu, Anna Di Gregorio
Ascidian embryos have been employed as model systems for studies of developmental biology for well over a century, owing to their desirable blend of experimental advantages, which include their rapid development, traceable cell lineage, and evolutionarily conserved morphogenetic movements. Two decades ago, the development of a streamlined electroporation method drastically reduced the time and cost of transgenic experiments, and, along with the elucidation of the complete genomic sequences of several ascidian species, propelled these simple chordates to the forefront of the model organisms available for studies of regulation of gene expression...
2018: Advances in Experimental Medicine and Biology
Robert W Zeller
Embryonic development depends on the orchestration of hundreds of regulatory and structural genes to initiate expression at the proper time, in the correct spatial domain(s), and in the amounts required for cells and tissues to become specified, determined, and ultimately to differentiate into a multicellular embryo. One of the key approaches to studying embryonic development is the generation of transgenic animals in which recombinant DNA molecules are transiently or stably introduced into embryos to alter gene expression, to manipulate gene function or to serve as reporters for specific cell types or subcellular compartments...
2018: Advances in Experimental Medicine and Biology
Kenji Kobayashi, Yutaka Satou
Microinjection is a common technique used to deliver nucleic acids into eggs and embryos in Ciona species. There are three Ciona species that are commonly used for research-Ciona intestinalis type A (C. robusta), C. intestinalis type B (C. intestinalis), and C. savignyi. Here, we present the microinjection methods using eggs and embryos of C. intestinalis type A and C. savignyi; however, our methods would also be applicable to eggs and embryos of C. intestinalis type B. Microinjection is a classical and widely used delivery method, which involves the use of a glass micropipette, a hollow glass needle with a microscopic tip, to inject nucleic acids into eggs and embryos under a stereo microscope...
2018: Advances in Experimental Medicine and Biology
Marie Teixeira, Bénédicte F Py, Christophe Bosc, Daphné Laubreton, Marie-Jo Moutin, Jacqueline Marvel, Frédéric Flamant, Suzy Markossian
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
March 13, 2018: Scientific Reports
M Fiorentzis, H Kalirai, P Katopodis, B Seitz, A Viestenz, S E Coupland
Electrochemotherapy (ECT) enhances responsiveness to cytotoxic drugs in numerous cell lines in vitro. Clinically ECT is widely applied for skin tumor ablation and has shown efficacy in treating non-resectable colorectal liver metastases. There is limited experience of ECT for ocular tumor therapy. We investigated the cytotoxic effect of bleomycin and cisplatin in combination with electroporation on chemoresistant human uveal melanoma (UM) cell lines in vitro. Four UM cell lines (Mel 270, 92-1, OMM-1, OMM-2...
2018: Neoplasma
Agnete B Madsen, Jonas R Knudsen, Carlos Henriquez-Olguin, Yeliz Angin, Kristien J Zaal, Lykke Sylow, Peter Schjerling, Evelyn Ralston, Thomas E Jensen
Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the β-actin isoform which, in many cell types, is the main actin isoform. However, it is not clear that β-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on β-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of β-actin in fully differentiated muscle, we have performed a detailed characterization of wildtype and muscle-specific β-actin knockout (KO) mice...
March 13, 2018: American Journal of Physiology. Endocrinology and Metabolism
Imran A Siddiqui, Amit V Sastry, John B Martinie, Dionisios Vrochides, Erin H Baker, David A Iannitti
BACKGROUND: Sixteen hepatopancreatobiliary fellowship programs in North America are accredited by the Fellowship Council. This study aims to assess fellows' perceptions of their training program. METHODS: A multiple-choice questionnaire was sent to 35 fellows to assess how they perceived their training: academics, research, operative experiences, autonomy, mentorship, program quality and weaknesses. The survey was developed using the SurveyMonkey® tool. RESULTS: Twenty-four of 35 fellows completed the survey...
March 7, 2018: HPB: the Official Journal of the International Hepato Pancreato Biliary Association
C Burkart, A Mukhopadhyay, S A Shirley, R J Connolly, J H Wright, A Bahrami, J S Campbell, R H Pierce, D A Canton
The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines results in serious, potentially life-threatening, adverse effects. By contrast, nucleic acid transfer via electroporation (EP) is a safe and effective method of delivering plasmid-encoded cytokines to tumors. Intratumoral delivery of IL-12 plasmid DNA by electroporation (IT-pIL12-EP) produced objective response rates in Phase 2 clinical trials in metastatic melanoma...
March 9, 2018: Gene Therapy
Xianchan Li, Lin Ren, Johan Dunevall, Daixin Ye, Henry S White, Martin A Edwards, Andrew G Ewing
The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modelling to calculate this collection efficiency...
March 7, 2018: ACS Nano
Haixia Zhang, Kuangpeng Liu, Zhixiao Xue, Huijuan Yin, Huajiang Dong, Wendong Jin, Xiafei Shi, Han Wang, Hai Wang
This study evaluated the effects and mechanism of action of combining irreversible electroporation (IRE) and photodynamic therapy (PDT) in breast cancer cells in vitro and in vivo . Jin's formula was used to assess killing efficacy of different IRE+PDT dosing combinations in breast cancer MCF-7 cells. Flow cytometry, high-content imaging, and confocal laser scanning microscopy were used to detect apoptosis. qRT-PCR and western blotting were used to evaluate expression of apoptosis-related genes and proteins...
2018: American Journal of Translational Research
Young Hong, Jonathan Rice, Divyansh Sharma, Robert C G Martin
INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) often presents late with only 20% of patients being candidates for resection while majority already have advanced metastases with median overall survival of 3-6 months. Currently, the role of oligometastasectomy and local therapy options in PDAC is unknown in patients who have favorable response to systemic chemotherapy. The aim of this study is to analyze the survival outcome of oligometastasectomy and local IRE therapy in select patients who are treated with systemic chemotherapy for PDAC metastases...
March 2, 2018: American Journal of Surgery
Hai-Tao Hou, Jin-Yang Lv, Zhi-Qiang Zhang, Ying Lu, Cui-Ping Zhou, Tian-Qiu Zhou, Shu-Qiang Zhang, Hui Xu
In vivo electroporation of morpholinos (MOs) into the retina of adult zebrafish is an efficient method to study gene function related to retinal disease and regeneration. However, the currently reported methods are complicated with low MO transfer efficiency and high probability to cause collateral damage. The present study was aimed to optimize the existing MO electroporation methods. Two major changes were made to MO electroporation procedure in zebrafish retina. One was to coat the inner side of the electrode with ultrasonic gel...
February 25, 2018: Sheng Li Xue Bao: [Acta Physiologica Sinica]
Evelin Kámán-Tóth, Miklós Pogány, Tamás Dankó, Ágnes Szatmári, Zoltán Bozsó
Agrobacterium tumefaciens is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent Agrobacterium cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures...
March 2018: 3 Biotech
Kamil R Wierzbinski, Tomasz Szymanski, Natalia Rozwadowska, Jakub D Rybka, Agnieszka Zimna, Tomasz Zalewski, Karolina Nowicka-Bauer, Agnieszka Malcher, Magdalena Nowaczyk, Michal Krupinski, Michal Fiedorowicz, Piotr Bogorodzki, Pawel Grieb, Michal Giersig, Maciej K Kurpisz
Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction...
February 27, 2018: Scientific Reports
Michael B Sano, Matthew R DeWitt, Stephanie D Teeter, Lei Xing
High-frequency irreversible electroporation (H-FIRE) is an emerging ablation modality, delivering rapid bursts of bipolar microsecond-duration electrical pulses to non-thermally ablate tissue including tumors. With advantages over current electroporation techniques including mitigation of muscle stimulation and reduced susceptibility to heterogeneous tissue properties, H-FIRE may produce more uniform and predictable ablations and can potentially be delivered with a single applicator device. However, the resulting ablations tend to be smaller than those provided with equivalent energy monopolar pulse protocols...
February 17, 2018: Computers in Biology and Medicine
Masato Ohtsuka, Masahiro Sato, Hiromi Miura, Shuji Takabayashi, Makoto Matsuyama, Takayuki Koyano, Naomi Arifin, Shingo Nakamura, Kenta Wada, Channabasavaiah B Gurumurthy
We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise...
February 26, 2018: Genome Biology
Honghao Yu, Weihu Long, Xuezeng Zhang, Kaixiang Xu, Jianxiong Guo, Heng Zhao, Honghui Li, Yubo Qing, Weirong Pan, Baoyu Jia, Hong-Ye Zhao, Xingxu Huang, Hong-Jiang Wei
BACKGROUND: Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome...
February 27, 2018: Journal of Translational Medicine
Li Tian, Yang Qiao, Patrick Lee, Lucas Wang, Ashley Chang, Saisree Ravi, Thomas A Rogers, Linfeng Lu, Burapol Singhana, Jun Zhao, Marites P Melancon
Irreversible electroporation (IRE) is an emerging minimally invasive tumor ablation technique that delivers short pulses of strong electric fields and kills cancer cells by disrupting their cell membranes with the electric pulses. However, clinical studies report that more than 10% of local tumor recurrences occur at the original ablated site. NVP BEZ-235 (BEZ) is a dual PI3K/mTOR inhibitor that has substantial anticancer effects. However, the clinical trials of BEZ was not satisfactory because of its low bioavailability and high toxicity, which stemmed from the use of oral administration of high doses over a long period of time...
November 2018: Drug Delivery
Govindarajan Srimathveeravalli, Dalya Abdel-Atti, Carlos Pérez-Medina, Haruyuki Takaki, Stephen B Solomon, Willem J M Mulder, Thomas Reiner
Reversible electroporation (RE) can facilitate nanoparticle delivery to tumors through direct transfection and from changes in vascular permeability. We investigated a radiolabeled liposomal nanoparticle (89 Zr-NRep) for monitoring RE-mediated liposomal doxorubicin (DOX) delivery in mouse tumors. Intravenously delivered89 Zr-NRep allowed positron emission tomography imaging of electroporation-mediated nanoparticle uptake. The relative order of89 Zr-NRep injection and electroporation did not result in significantly different overall tumor uptake, suggesting direct transfection and vascular permeability can independently mediate deposition of89 Zr-NRep in tumors...
January 2018: Molecular Imaging
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