Mechanistic studies of gene delivery into mammalian cells by electrical short-circuiting via an aqueous droplet in dielectric oil

We have developed a novel methodology for the delivery of cell-impermeable molecules, based on electrical short-circuiting via a water droplet in dielectric oil. When a cell suspension droplet is placed between a pair of electrodes with an intense DC electric field, droplet bouncing and droplet deformation, which results in an instantaneous short-circuit, can be induced, depending on the electric field strength. We have demonstrated successful transfection of various mammalian cells using the short-circuiting; however, the molecular mechanism remains to be elucidated. In this study, flow cytometric assays were performed with Jurkat cells. An aqueous droplet containing Jurkat cells and plasmids carrying fluorescent proteins was treated with droplet bouncing or short-circuiting. The short-circuiting resulted in sufficient cell viability and fluorescent protein expression after 24 hours’ incubation. In contrast, droplet bouncing did not result in successful gene transfection. Transient membrane pore formation was investigated by uptake of a cell-impermeable fluorescence dye YO-PRO-1 and the influx of calcium ions. As a result, short-circuiting increased YO-PRO-1 fluorescence intensity and intracellular calcium ion concentration, but droplet bouncing did not. We also investigated the contribution of endocytosis to the transfection. The pre-treatment of cells with endocytosis inhibitors decreased the efficiency of gene transfection in a concentration-dependent manner. Besides, the use of pH-sensitive dye conjugates indicated the formation of an acidic environment in the endosomes after the short-circuiting. Endocytosis is a possible mechanism for the intracellular delivery of exogenous DNA.

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Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

Infection and Immunity ◽

10.1128/iai.74.5.2937-2946.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2937-2946 ◽

Cited By ~ 27

Author(s):

A. Ghosh ◽

D. R. Saha ◽

K. M. Hoque ◽

M. Asakuna ◽

S. Yamasaki ◽

...

Keyword(s):

Cholera Toxin ◽

Vibrio Cholerae ◽

Hela Cells ◽

Histopathological Examination ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Toxin Gene ◽

Dependent Manner ◽

A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

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Tracking Lysosome Migration within Chinese Hamster Ovary (CHO) Cells Following Exposure to Nanosecond Pulsed Electric Fields

Bioengineering ◽

10.3390/bioengineering5040103 ◽

2018 ◽

Vol 5 (4) ◽

pp. 103

Author(s):

Gary Thompson ◽

Hope Beier ◽

Bennett Ibey

Keyword(s):

Electric Field ◽

Electric Fields ◽

Mammalian Cells ◽

Plasma Membranes ◽

Chinese Hamster ◽

Lateral Diffusion ◽

Cell Swelling ◽

Dependent Manner ◽

Membrane Repair ◽

Osmotic Swelling

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.

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Motility patterns of Trypanosoma cruzi trypomastigotes correlate with the efficiency of parasite invasion in vitro

Scientific Reports ◽

10.1038/s41598-020-72604-4 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Jorge A. Arias-del-Angel ◽

Jesús Santana-Solano ◽

Moisés Santillán ◽

Rebeca G. Manning-Cela

Keyword(s):

Trypanosoma Cruzi ◽

Cell Line ◽

Mammalian Cells ◽

Dependent Manner ◽

Parasite Invasion ◽

Mammalian Cell Cultures ◽

A Cell ◽

Parasite Replication ◽

Parasite Motility

Abstract Numerous works have demonstrated that trypanosomatid motility is relevant for parasite replication and sensitivity. Nonetheless, although some findings indirectly suggest that motility also plays an important role during infection, this has not been extensively investigated. This work is aimed at partially filling this void for the case of Trypanosoma cruzi. After recording swimming T. cruzi trypomastigotes (CL Brener strain) and recovering their individual trajectories, we statistically analyzed parasite motility patterns. We did this with parasites that swim alone or above monolayer cultures of different cell lines. Our results indicate that T. cruzi trypomastigotes change their motility patterns when they are in the presence of mammalian cells, in a cell-line dependent manner. We further performed infection experiments in which each of the mammalian cell cultures were incubated for 2 h together with trypomastigotes, and measured the corresponding invasion efficiency. Not only this parameter varied from cell line to cell line, but it resulted to be positively correlated with the corresponding intensity of the motility pattern changes. Together, these results suggest that T. cruzi trypomastigotes are capable of sensing the presence of mammalian cells and of changing their motility patterns accordingly, and that this might increase their invasion efficiency.

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Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0457 ◽

2011 ◽

Vol 22 (24) ◽

pp. 4822-4833 ◽

Cited By ~ 58

Author(s):

Vinay Ramabhadran ◽

Farida Korobova ◽

Gilbert J. Rahme ◽

Henry N. Higgs

Keyword(s):

Actin Filaments ◽

Splice Variants ◽

Cellular Function ◽

Jurkat Cells ◽

Dependent Manner ◽

Latrunculin B ◽

Independent Manner ◽

3T3 Fibroblasts ◽

Golgi Region ◽

A Cell

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type–dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2–non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2–non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2–non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer–sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.

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The Ca2+-binding Protein ALG-2 Is Recruited to Endoplasmic Reticulum Exit Sites by Sec31A and Stabilizes the Localization of Sec31A

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0444 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4876-4887 ◽

Cited By ~ 72

Author(s):

Akinori Yamasaki ◽

Katsuko Tani ◽

Akitsugu Yamamoto ◽

Naomi Kitamura ◽

Masayuki Komada

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Binding Protein ◽

Small Gtpase ◽

Dependent Manner ◽

Rich Region ◽

Linked Gene ◽

Transport Vesicles ◽

Er Exit Sites ◽

A Cell

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca2+-dependent manner and colocalized with Sec31A at ER exit sites. A Ca2+binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca2+chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca2+-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

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Codon bias determines sorting of ion channel protein

10.1101/2020.03.17.994780 ◽

2020 ◽

Author(s):

Marina Kithil ◽

Anja Jeannine Engel ◽

Markus Langhans ◽

Oliver Rauh ◽

Matea Cartolano ◽

...

Keyword(s):

Mammalian Cells ◽

Secretory Pathway ◽

Model Systems ◽

Dependent Manner ◽

Intracellular Proteins ◽

Polypeptide Folding ◽

A Cell ◽

Identical Amino Acid Sequence ◽

Cycle Dependent ◽

The Impact

AbstractThe choice of codons can influence local translation kinetics during protein synthesis. The question of whether the modulation of polypeptide folding and binding to chaperons influences sorting of nascent membrane proteins remains unclear. Here, we use two similar K+ channels as model systems to examine the impact of codon choice on protein sorting. By monitoring transient expression of GFP tagged proteins in mammalian cells we find that targeting of one channel to the secretory pathway is insensitive to codon optimization. In contrast, sorting of the second channel to the mitochondria is very sensitive to codon choice. The protein with an identical amino acid sequence is sorted in a codon and cell cycle dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves together with a cell-state depending decoding mechanism as a secondary code for sorting intracellular proteins.

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Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of genetic payloads for rapid cell engineering

10.1101/2020.03.25.008276 ◽

2020 ◽

Author(s):

Darin Bloemberg ◽

Daniela Sosa-Miranda ◽

Tina Nguyen ◽

Risini D. Weeratna ◽

Scott McComb

Keyword(s):

T Cell Activation ◽

Mammalian Cells ◽

Cell Activation ◽

Genome Engineering ◽

Jurkat Cells ◽

Sequencing Analysis ◽

Knock Out ◽

Homology Directed Repair ◽

A Cell ◽

Future Utility

AbstractSince observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited indel formation. Interestingly, including longer homology arms (HAs) (500 bp) in varying orientations only modestly increased knock-in efficiency (∼2-fold). Using a SCIP-payload design (SCIPpay) which liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair (HDR) for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs-of-concept, we used SCIPpay to 1) insert a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T cell activation, and 2) insert a chimeric antigen receptor (CAR) gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines and suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.

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Regulation of the expression and activity of Unr in mammalian cells

Biochemical Society Transactions ◽

10.1042/bst20150165 ◽

2015 ◽

Vol 43 (6) ◽

pp. 1241-1246 ◽

Cited By ~ 15

Author(s):

Emma C. Anderson ◽

Pól Ó Catnaigh

Keyword(s):

Mammalian Cells ◽

Alternative Polyadenylation ◽

Dependent Manner ◽

Factor X ◽

Mammalian Development ◽

Ras Gene ◽

Polypyrimidine Tract Binding Protein ◽

A Cell ◽

Internal Initiation ◽

Cycle Dependent

Unr (upstream of N-ras) is a post-transcriptional regulator of gene expression, essential for mammalian development and mutated in many human cancers. The expression of unr is itself regulated at many levels; transcription of unr, which also affects expression of the downstream N-ras gene, is tissue and developmental stage-dependent and is repressed by c-Myc and Max (Myc associated factor X). Alternative splicing gives rise to six transcript variants, which include three different 5′-UTRs. The transcripts are further diversified by the use of three alternative polyadenylation signals, which governs whether AU-rich instability elements are present in the 3′-UTR or not. Translation of at least some unr transcripts can occur by internal initiation and is regulated in a cell-cycle-dependent manner; binding of PTB (polypyrimidine tract-binding protein) and Unr to the 5′-UTR inhibits translation, but these are displaced by heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNPC1/C2) during mitosis to stimulate translation. Finally, Unr is post-translationally modified by phosphorylation and lysine acetylation, although it is not yet known how these modifications affect Unr activity.

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Improvement of mRNA Delivery Efficiency to a T Cell Line by Modulating PEG-Lipid Content and Phospholipid Components of Lipid Nanoparticles

Pharmaceutics ◽

10.3390/pharmaceutics13122097 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2097

Author(s):

Hiroki Tanaka ◽

Ryo Miyama ◽

Yu Sakurai ◽

Shinya Tamagawa ◽

Yuta Nakai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Messenger Rna ◽

Gene Transfection ◽

Lipid Nanoparticles ◽

Jurkat Cells ◽

Target Antigen ◽

Target Cells ◽

Exogenous Dna

(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.

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