scholarly journals Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1492
Author(s):  
Sabina Sánchez-Hernández ◽  
Araceli Aguilar-González ◽  
Beatriz Guijarro-Albaladejo ◽  
Noelia Maldonado-Pérez ◽  
Iris Ramos-Hernández ◽  
...  
Keyword(s):  
Cell Lines ◽  
Main Concern ◽  
Target Sequence ◽  
Gene Repair ◽  
Reporter Cell ◽  
Delivery Method ◽  
Cellular Models ◽  
Target Sites ◽  
Reporter Systems ◽  
Or Gene

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.

scholarly journals Cell Cultures for Virology: Usability, Advantages, and Prospects

2020 ◽  
Vol 21 (21) ◽  
pp. 7978
Author(s):  
Alexander A. Dolskiy ◽  
Irina V. Grishchenko ◽  
Dmitry V. Yudkin
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Virus Isolation ◽  
Virus Detection ◽  
Clinical Samples ◽  
Laboratory Setting ◽  
Reporter Cell ◽  
Isolation And Identification ◽  
Reporter Systems ◽  
Reporter Proteins

Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.

scholarly journals Genome-Engineering Tools to Establish Accurate Reporter Cell Lines That Enable Identification of Therapeutic Strategies to Treat Friedreich’s Ataxia

2015 ◽  
Vol 20 (6) ◽  
pp. 760-767
Author(s):  
Rodrigo Villaseñor ◽  
Loren Miraglia ◽  
Angelica Romero ◽  
Buu Tu ◽  
Tanel Punga ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Genome Engineering ◽  
Friedreich’S Ataxia ◽  
Friedreich's Ataxia ◽  
Luciferase Reporter ◽  
Reporter Cell ◽  
Cellular Models ◽  
Frataxin Gene

Friedreich’s ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genome-engineering tools that enable the generation of human cell lines that express the frataxin gene fused to a luciferase reporter gene from its endogenous locus. Performing a pilot high-throughput genomic screen in a newly established reporter cell line, we uncovered novel negative regulators of frataxin expression. Rational design of small-molecule inhibitors of the identified frataxin repressors and/or high-throughput screening of large siRNA or compound libraries with our system may yield treatments for Friedreich’s ataxia.

scholarly journals HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Giuliana Mastropietro ◽  
Inés Tiscornia ◽  
Karen Perelmuter ◽  
Soledad Astrada ◽  
Mariela Bollati-Fogolín
Keyword(s):  
Cell Lines ◽  
Nuclear Factor Kappa B ◽  
Biological Processes ◽  
Bacterial Strains ◽  
Reporter Cell ◽  
Functional Studies ◽  
Primary Screening ◽  
Screening And Identification ◽  
Reporter Systems ◽  
Ht 29

The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

Pharmacological characterization of receptor guanylyl cyclase reporter cell lines

2013 ◽  
Vol 698 (1-3) ◽  
pp. 131-136 ◽  
Author(s):  
Frank Wunder ◽  
Annette Woermann ◽  
Andreas Geerts ◽  
Markus Milde
Keyword(s):  
Cell Lines ◽  
Guanylyl Cyclase ◽  
Reporter Cell ◽  
Pharmacological Characterization

scholarly journals Generation of Double-Labeled Reporter Cell Lines for Studying Co-Dynamics of Endogenous Proteins in Individual Human Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (10) ◽  
pp. e13524 ◽  
Author(s):  
Irina Issaeva ◽  
Ariel A. Cohen ◽  
Eran Eden ◽  
Cellina Cohen-Saidon ◽  
Tamar Danon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cells ◽  
Reporter Cell ◽  
Endogenous Proteins ◽  
Individual Human

scholarly journals Pharmacological characterization of receptor guanylyl cyclase reporter cell lines

2009 ◽  
Vol 9 (S1) ◽  
Author(s):  
Frank Wunder ◽  
Daniel Barufe ◽  
Annette Woermann ◽  
Markus Milde
Keyword(s):  
Cell Lines ◽  
Guanylyl Cyclase ◽  
Reporter Cell ◽  
Pharmacological Characterization

Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands

Luminescence ◽  
2001 ◽  
Vol 16 (2) ◽  
pp. 153-158 ◽  
Author(s):  
Patrick Balaguer ◽  
Anne-Marie Boussioux ◽  
Ediz Demirpence ◽  
Jean-Claude Nicolas
Keyword(s):  
Biological Activity ◽  
Cell Lines ◽  
Nuclear Receptor ◽  
Receptor Ligands ◽  
Reporter Cell ◽  
Nuclear Receptor Ligands

scholarly journals Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro

2014 ◽  
Vol 13 (2) ◽  
pp. 251-261 ◽  
Author(s):  
Dmitry A. Ovchinnikov ◽  
Drew M. Titmarsh ◽  
Patrick R.J. Fortuna ◽  
Alejandro Hidalgo ◽  
Samah Alharbi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Reporter Cell ◽  
Selection Of

scholarly journals Differential Regulation of Telomeric Complex by BCR-ABL1 Kinase in Human Cellular Models of Chronic Myeloid Leukemia—From Single Cell Analysis to Next-Generation Sequencing

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1145
Author(s):  
Anna Deregowska ◽  
Monika Pepek ◽  
Katarzyna Pruszczyk ◽  
Marcin M. Machnicki ◽  
Maciej Wnuk ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Chronic Myeloid Leukemia ◽  
Telomere Length ◽  
Cell Lines ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Telomere Maintenance ◽  
Next Generation ◽  
Cellular Models ◽  
Generation Sequencing

Telomeres are specialized nucleoprotein complexes, localized at the physical ends of chromosomes, that contribute to the maintenance of genome stability. One of the features of chronic myeloid leukemia (CML) cells is a reduction in telomere length which may result in increased genomic instability and progression of the disease. Aberrant telomere maintenance in CML is not fully understood and other mechanisms such as the alternative lengthening of telomeres (ALT) are involved. In this work, we employed five BCR-ABL1-positive cell lines, namely K562, KU-812, LAMA-84, MEG-A2, and MOLM-1, commonly used in the laboratories to study the link between mutation, copy number, and expression of telomere maintenance genes with the expression, copy number, and activity of BCR-ABL1. Our results demonstrated that the copy number and expression of BCR-ABL1 are crucial for telomere lengthening. We observed a correlation between BCR-ABL1 expression and telomere length as well as shelterins upregulation. Next-generation sequencing revealed pathogenic variants and copy number alterations in major tumor suppressors, such as TP53 and CDKN2A, but not in telomere-associated genes. Taken together, we showed that BCR-ABL1 kinase expression and activity play a crucial role in the maintenance of telomeres in CML cell lines. Our results may help to validate and properly interpret results obtained by many laboratories employing these in vitro models of CML.

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