scholarly journals Use of the Site-Specific Retargeting Jump-In Platform Cell Line to Support Biologic Drug Discovery

2014 ◽  
Vol 20 (4) ◽  
pp. 528-535 ◽  
Author(s):  
Robin Butler ◽  
David Hornigold ◽  
Ling Huang ◽  
Catherine Huntington ◽  
Tim London ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Cell Line ◽  
Cell Lines ◽  
Stable Cell Line ◽  
Therapeutic Antibodies ◽  
Site Specific ◽  
Mammalian Cell Lines ◽  
Cell To Cell Variability ◽  
High Degree

Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this article, we describe how the use of the Jump-In platform (Life Technologies, Carlsbad, CA) has been applied to drug discovery projects. It can easily and rapidly generate homogeneous high-expressing cell pools with a high degree of reproducibility. Their use in cell-based screening to identify specific binders, identify binding to relevant species variants, or detect functionally relevant therapeutic antibodies is central in driving drug discovery.

scholarly journals ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS

1973 ◽  
Vol 58 (1) ◽  
pp. 11-26 ◽  
Author(s):  
Wolfgang Klietmann ◽  
Nobuhiro Sato ◽  
Margit M. K. Nass
Keyword(s):  
Mitochondrial Dna ◽  
Cell Line ◽  
Cell Lines ◽  
Ethidium Bromide ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Ultrastructural Changes ◽  
Parental Cell Line ◽  
Mammalian Cell Lines

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a3 show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.

scholarly journals Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

2021 ◽  
Author(s):  
Shuiyun Lan ◽  
Philip R Tedbury ◽  
Yee Tsuey Ong ◽  
Raven Shah ◽  
Ryan L Slack ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Cell Line ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Stable Cell Line ◽  
Target Cells ◽  
Mechanistic Studies ◽  
Artificial Chromosomes ◽  
Novel Approach

Replicon-based technologies were used to develop reagents and assays for advanced drug discovery efforts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for examining all facets of the SARS-CoV-2 replication cycle at reduced biocontainment level. Specifically: a) 21 replicons were cloned in bacterial artificial chromosomes (BACs) and delivered as transfectable plasmid DNA or transcribed RNA in various cell types. Replicons carrying mutations that affect the activity or antiviral susceptibility of SARS-CoV-2 enzymes were used to establish utility for mechanistic studies while reducing the community risks associated with gain-of-function studies in fully infectious virus. b) A BHK-21 stable cell line harboring SARS-CoV-2 replicon was generated and characterized in robust high/ultra-high throughput assays of antiviral efficacy with orthogonal SARS-CoV-2 replication reporter genes (Nano luciferase and enhanced green fluorescent protein-eGFP); the estimated antiviral potencies in the fully infectious SARS-CoV-2 system and in the transient or stable replicon systems were similar. HEK293 and Calu1 stable cell lines expressing SARS-CoV-2 replicon have also been prepared. Finally, c) we generated trans-encapsidated replicons by co-expression with SARS-CoV-2 structural proteins, thus producing single-round infectious SARS-CoV-2 virus-like particles able to transduce susceptible cell types, thus expanding utility to enable study of virion assembly and entry into target cells. Hence, these SARS-CoV-2 replicon-based reagents include a novel approach to replicon-harboring cell line generation and are valuable tools that can be used at lower biosafety level (BSL2) for drug discovery efforts, characterization of SARS-CoV-2 and variant evolution in the COVID-19 pandemic, mechanisms of inhibition and resistance, and studies on the role of SARS-CoV-2 genes and host dependency factors.

Establishment and characterization of 7 ovarian carcinoma cell lines and one granulosa tumor cell line: Growth features and cytogenetics

1993 ◽  
Vol 53 (4) ◽  
pp. 613-620 ◽  
Author(s):  
Cornelia A. M. van den Berg-Bakker ◽  
Anne Hagemeijer ◽  
Elsa M. Franken-Postma ◽  
Vincent T. H. B. M. Smit ◽  
Peter J. K. Kuppen ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Ovarian Carcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Tumor Cell Line ◽  
Carcinoma Cell Lines ◽  
Growth Features

Quantitative cell fusion: the fusion sensitivity (FS) potential

1981 ◽  
Vol 49 (1) ◽  
pp. 87-97
Author(s):  
D. Rohme
Keyword(s):  
Cell Line ◽  
Cell Fusion ◽  
Cell Lines ◽  
Dose Response ◽  
Sendai Virus ◽  
Biological Significance ◽  
Diploid Cell ◽  
Mammalian Cell Lines ◽  
Virus Dose ◽  
A Cell

The dose response of Sendai virus-induced cell fusion was studied in 10 mammalian cell lines, comprising 5 continuous and 5 diploid cell lines originating from 5 species. The extent of fusion was calculated using a parameter directly proportional to the number of fusion events (t-parameter). At lower levels of fusion the dose response was found to be based on the same simple kinetic rules in all cell lines and was defined by the formula: t = FS. FAU/(I + FS. FAU), where FS (fusion sensitivity) is a cell-specific constant of the fusion rate and FAU (fusion activity units) is the virus dose. The FS potential of a cell line was determined as the linear regression coefficient of the fusion index (t/(I - t)) on the virus dose. At higher levels of fusion, when the fusion extent reached cell-line-specific maximal levels, the dose response was not as uniform. In general, and particularly in the cases of the diploid cell lines, these maximal levels were directly proportional to the FS potentials. Thus, it was concluded that the FS potential is the basic quantitative feature, which expresses the cellular fusion efficiency. The fact that FS varied extensively between cell lines, but at the same time apparently followed certain patterns (being higher in continuous compared to diploid cell lines and being related to the species of origin of the cells), emphasizes it biological significance as well as its possible usefulness in studies of the efficiency of various molecular interactions in the cell membrane/cytoskeleton system.

Characterization of cell-banking parameters for the cryopreservation of mammalian cell lines in 100-mL cryobags

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
Rüdiger Heidemann ◽  
Svenja Lünse ◽  
Doan Tran ◽  
Chun Zhang
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cell Lines ◽  
Cell Banking

scholarly journals Drug Discovery for Chagas Disease: Impact of Different Host Cell Lines on Assay Performance and Hit Compound Selection

2019 ◽  
Vol 4 (2) ◽  
pp. 82 ◽  
Author(s):  
Caio Haddad Franco ◽  
Laura Maria Alcântara ◽  
Eric Chatelain ◽  
Lucio Freitas-Junior ◽  
Carolina Borsoi Moraes
Keyword(s):  
Drug Discovery ◽  
Host Cell ◽  
Cell Lines ◽  
Chagas Disease ◽  
Host Cells ◽  
Mammalian Cell Lines ◽  
Starting Point ◽  
Disease Impact ◽  
Screening Assays ◽  
Host Parasite

Cell-based screening has become the major compound interrogation strategy in Chagas disease drug discovery. Several different cell lines have been deployed as host cells in screening assays. However, host cell characteristics and host-parasite interactions may play an important role when assessing anti-T. cruzi compound activity, ultimately impacting on hit discovery. To verify this hypothesis, four distinct mammalian cell lines (U2OS, THP-1, Vero and L6) were used as T. cruzi host cells in High Content Screening assays. Rates of infection varied greatly between different host cells. Susceptibility to benznidazole also varied, depending on the host cell and parasite strain. A library of 1,280 compounds was screened against the four different cell lines infected with T. cruzi, resulting in the selection of a total of 82 distinct compounds as hits. From these, only two hits were common to all four cell lines assays (2.4%) and 51 were exclusively selected from a single assay (62.2%). Infected U2OS cells were the most sensitive assay, as 55 compounds in total were identified as hits; infected THP-1 yielded the lowest hit rates, with only 16 hit compounds. Of the selected hits, compound FPL64176 presented selective anti-T. cruzi activity and could serve as a starting point for the discovery of new anti-chagasic drugs.

scholarly journals Cell Line Classification Using Electric Cell-Substrate Impedance Sensing (ECIS)

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Megan L. Gelsinger ◽  
Laura L. Tupper ◽  
David S. Matteson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Statistical Tests ◽  
Classification Methods ◽  
Impedance Sensing ◽  
Mammalian Cell Lines ◽  
Cell Substrate ◽  
Multiple Cell ◽  
Line Classification

AbstractWe present new methods for cell line classification using multivariate time series bioimpedance data obtained from electric cell-substrate impedance sensing (ECIS) technology. The ECIS technology, which monitors the attachment and spreading of mammalian cells in real time through the collection of electrical impedance data, has historically been used to study one cell line at a time. However, we show that if applied to data from multiple cell lines, ECIS can be used to classify unknown or potentially mislabeled cells, factors which have previously been associated with the reproducibility crisis in the biological literature. We assess a range of approaches to this new problem, testing different classification methods and deriving a dictionary of 29 features to characterize ECIS data. Most notably, our analysis enriches the current field by making use of simultaneous multi-frequency ECIS data, where previous studies have focused on only one frequency; using classification methods to distinguish multiple cell lines, rather than simple statistical tests that compare only two cell lines; and assessing a range of features derived from ECIS data based on their classification performance. In classification tests on fifteen mammalian cell lines, we obtain very high out-of-sample predictive accuracy. These preliminary findings provide a baseline for future large-scale studies in this field.

scholarly journals Domain-invariant features for mechanism of action prediction in a multi-cell-line drug screen

2019 ◽  
Vol 36 (5) ◽  
pp. 1607-1613 ◽  
Author(s):  
Joseph C Boyd ◽  
Alice Pinheiro ◽  
Elaine Del Nery ◽  
Fabien Reyal ◽  
Thomas Walter
Keyword(s):  
Drug Discovery ◽  
Single Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Molecular Subtype ◽  
Supplementary Information ◽  
Breast Cancer Cell Lines ◽  
Molecular Heterogeneity ◽  
Drug Mechanism

Abstract Motivation High-content screening is an important tool in drug discovery and characterization. Often, high-content drug screens are performed on one single-cell line. Yet, a single-cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterize drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process. Results The contribution of this article is 2-fold. First, we investigate whether we can predict drug mechanism of action (MOA) at the molecular level without optimization of the MOA classes to the screen specificities. To this end, we benchmark a set of algorithms within a conventional pipeline, and evaluate their MOA prediction performance according to a statistically rigorous framework. Second, we extend this conventional pipeline to the simultaneous analysis of multiple cell lines, each manifesting potentially different morphological baselines. For this, we propose multi-task autoencoders, including a domain-adaptive model used to construct domain-invariant feature representations across cell lines. We apply these methods to a pilot screen of two triple negative breast cancer cell lines as models for two different molecular subtypes of the disease. Availability and implementation https://github.com/jcboyd/multi-cell-line or https://zenodo.org/record/2677923. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Site-specific N-glycosylation of HeLa cell glycoproteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lilla Turiák ◽  
Simon Sugár ◽  
András Ács ◽  
Gábor Tóth ◽  
Ágnes Gömöry ◽  
...  
Keyword(s):  
Hela Cell ◽  
Cell Line ◽  
Bovine Serum ◽  
Culture Media ◽  
Cancer Cell Line ◽  
Hela Cell Line ◽  
Glycosylation Pattern ◽  
Site Specific ◽  
Glycosylation Sites

Abstract We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media – nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.

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