Cloning and Selection

Freshney, R. Ian

doi:10.1002/0471747599.cac014

Cited by 3 publications

(3 citation statements)

References 56 publications

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“…An alternative and commonly used method to achieve single cell cloning is by using fluorescence-activated cell sorting (FACS) [39] or, less frequently, more sophisticated technologies such as Laser Capture Microdissection (LCM) [40], microfluidic devices [41,42] and Magnetic-Activated Cell Sorting (MACS) [43]. Before the advent of automated single cell isolation methods, the use of cloning cylinders, together with the limiting dilution method, represented one of the most important approaches for the production of clonal cell lines [44][45][46][47].This approach has been recently used for the establishment of human multipotent progenitor cells [48] and the isolation CRISPR edited mammalian cell lines [49]. However, single clone isolation by means of cloning cylinders in fish cells has not yet been reported.…”

Section: Introductionmentioning

confidence: 99%

A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

et al. 2021

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Background The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. Results Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. Conclusions The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

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Section: Introductionmentioning

confidence: 99%

A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

et al. 2021

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“…We particularly picked up single cell which expressed in the epithelial-like cell morphology among these heterogeneous primary cell cultures using small paper filter soaked with digestive solution, 0.1% trypsin (Becton-Dickinson, Franklin Lakes, NJ, USA) and 0.02% ethylenediaminetetraacetic acid (EDTA; Dojindo, Kumamoto, Japan) in phosphate-buffered saline without calcium and magnesium (PBS[−]). This single cell isolation was a modified method from classical colony isolation by cloning ring technique [ 16 ]. The clonal cells were then isolated into 24-well plates (Nunc, Thermo Fisher Scientific, Pittsburgh, USA) and expanding to 60-mm 2 culture dishes (Nunc) after reached to 70–80% confluence in the well under 37 °C/4.7% CO 2 conditions.…”

Section: Methodsmentioning

confidence: 99%

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro

et al. 2017

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In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial–endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.Electronic supplementary materialThe online version of this article (doi:10.1007/s13577-017-0173-y) contains supplementary material, which is available to authorized users.

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“…In the most simple cell culture models, one single cell type is plated and consequently only homotypic signals are present. To compensate for the lack of heterotypic signals, serum or conditioned medium containing an assortment of growth factors, proteins, amino acids, and metabolites that would otherwise be supplied by other cell types, can be added to the cell culture medium [41]. However, the use of such supplements is hampered by the inherent batch-to-batch variability in concentration of minor components that can affect cell growth and function, resulting in variations between the same cell culture model [42].…”

Section: Cell-cell Communication and Co-culturesmentioning

confidence: 99%

Organs-on-chips for the pharmaceutical development process: design perspectives and implementations

Christoffersson¹

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Organs-on-chips are dynamic cell culture devices created with the intention to mimic organ function in vitro. Their purpose is to assess the toxicity and efficacy of drugs and, as early as possible in the pharmaceutical development process, predict the outcome of clinical trials. The aim of this thesis is to explain and discuss these cell culture devices from a design perspective and to experimentally exemplify some of the specific functions that characterize organs-on-chips.The cells in our body reside in complex environments with chemical and mechanical cues that affect their function and purpose. Such a complex environment is difficult to recreate in the laboratory and has therefore been overlooked in favor of more simple models, i.e. static twodimensional (2D) cell cultures. Numerous recent reports have shown cell culture systems that can resemble the cell's natural habitat and enhance cell functionality and thereby potentially provide results that better reflects animal and human trials. The way these organs-on-chips improve in vitro cell culture assays is to include e.g. a three-dimensional cell architecture (3D), mechanical stimuli, gradients of oxygen or nutrients, or by combining several relevant cell types that affect each other in close proximity.The research conducted for this thesis shows how cells in 3D spheroids or in 3D hydrogels can be cultured in perfused microbioreactors. Furthermore, a pump based on electroosmosis, and a method for an objective conceptual design process, is introduced to the field of organs-on-chips.Keywords: Organs-on-chips, cell culture models, pharmaceutical development, microfluidics iv v Populärvetenskaplig sammanfattningOrgans-on-chips är modellsystem med avsikt att återskapa ett organs funktion in vitro genom att odla celler från djur eller människor i en omgivning som efterliknar kroppen. Syftet med dessa modeller är att utvärdera ett potentiellt läkemedels toxicitet och effektivitet så tidigt som möjligt i läkemedelsutvecklingen för att kunna förutsäga hur det påverkar människor. Målet med denna avhandling är att förklara och diskutera denna typ av cellmodeller utifrån ett designperspektiv och experimentellt visa några av de specifika funktioner som är en del av konceptet organs-on-chips.Cellerna i våra kroppar befinner sig komplexa miljöer med kemiska och mekaniska signaler som påverkar deras funktion och syfte. Sådana miljöer är svåra att återskapa på laboratoriet och har därför till stor del förbisetts till fördel för enklare modellsystem som ofta består av celler som odlas på plast-eller glasytor under statiska förhållanden. På senare tid har en betydande mängd publikationer visat på att cellmodeller som bättre kan efterlikna cellens naturliga miljö kan förbättra dess olika funktioner och därmed ge resultat som eventuellt kan matcha försöken som görs på djur och människor. Det finns flera olika funktioner, hämtade från vår kunskap om människans biologi, som organs-on-chips kan implementera för att skapa dessa förbättrade cellmiljöer och inkluderar en tre-...

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Cloning and Selection

Cited by 3 publications

References 56 publications

A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro

Organs-on-chips for the pharmaceutical development process: design perspectives and implementations

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