scholarly journals Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency

2021 ◽  
Vol 49 (5) ◽  
pp. 2642-2654 ◽  
Author(s):  
Donghui Li ◽  
Xiaoji Sun ◽  
Fangzhou Yu ◽  
Mary Ann Perle ◽  
David Araten ◽  
...  
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Genome Engineering ◽  
Genome Project ◽  
Loss Of Function ◽  
Substantial Impact ◽  
Targeted Gene Deletion ◽  
Additional Cell ◽  
Selection Markers

Abstract The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).

scholarly journals Synchronization of human retinal pigment epithelial-1 cells in mitosis

2020 ◽  
Vol 133 (18) ◽  
pp. jcs247940
Author(s):  
Stacey J. Scott ◽  
Kethan S. Suvarna ◽  
Pier Paolo D'Avino
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Interaction Network ◽  
Protein Protein Interaction ◽  
Pigment Epithelial ◽  
Adenocarcinoma Cells ◽  
Sequential Treatments

ABSTRACTHuman retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80–90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib–nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein–protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

scholarly journals Synchronization of human retinal pigment ephitilial-1 (RPE-1) cells in mitosis

2020 ◽  
Author(s):  
Stacey J. Scott ◽  
Kethan Suvarna ◽  
Pier Paolo D’Avino
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Retinal Pigment ◽  
Interaction Network ◽  
Protein Protein Interaction ◽  
Adenocarcinoma Cells ◽  
Sequential Treatments ◽  
Dna Synthesis Inhibitors ◽  
Protein Protein Interaction Network

ABSTRACTHuman retinal pigment ephitilial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4/6 inhibitor PD 0332991 (palbociclib) and the microtubule depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Furthermore, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

scholarly journals Hi-C detects novel structural variants in HL-60 and HL-60/S4 cell lines

2018 ◽  
Author(s):  
Elsie C. Jacobson ◽  
Ralph S. Grand ◽  
Jo K. Perry ◽  
Mark H. Vickers ◽  
Ada L. Olins ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Rna Seq ◽  
Structural Variants ◽  
The Stability ◽  
Different Sources ◽  
Identification And Characterization

AbstractCancer cell lines often have large structural variants (SVs) that evolve over time. There are many reported differences in large scale SVs between HL-60 and HL-60/S4, two cell lines derived from the same acute myeloid leukemia sample. However, the stability and variability of inter- and intra-chromosomal structural variants between different sources of the same cell line is unknown. Here, we used Hi-C and RNA-seq to identify and compare large SVs in HL-60 and HL-60/S4 cell lines. Comparisons with previously published karyotypes identified novel SVs in both cell lines. Hi-C was used to characterize the known expansion centered on the MYC locus. The MYC expansion was integrated into known locations in HL-60/S4, and a novel location (chr4) in HL-60. The HL-60 cell line has more within-line structural variation than the HL-60/S4 derivative cell line. Collectively we demonstrate the usefulness of Hi-C and with RNA-seq data for the identification and characterization of SVs.

scholarly journals Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andrea Chai ◽  
Ana M Mateus ◽  
Fazal Oozeer ◽  
Rita Sousa-Nunes
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genetic Model ◽  
Model Organisms ◽  
Loss Of Function ◽  
Single Strain ◽  
Genetic Manipulations ◽  
Intrinsic Gene ◽  
Genetic Perturbations ◽  
Tool Set

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.

scholarly journals Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1312 ◽  
Author(s):  
Joffrey Mianné ◽  
Chloé Bourguignon ◽  
Chloé Nguyen Van ◽  
Mathieu Fieldès ◽  
Amel Nasri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Genome Engineering ◽  
Disease Modeling ◽  
Mutant Cell ◽  
Point Mutations ◽  
Human Pluripotent Stem Cells ◽  
Wide Range

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

scholarly journals Large-Scale Characterization of Drug Responses of Clinically Relevant Proteins in Cancer Cell Lines

Cancer Cell ◽  
2020 ◽  
Vol 38 (6) ◽  
pp. 829-843.e4
Author(s):  
Wei Zhao ◽  
Jun Li ◽  
Mei-Ju M. Chen ◽  
Yikai Luo ◽  
Zhenlin Ju ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Large Scale ◽  
Cancer Cell Lines ◽  
Drug Responses ◽  
Scale Characterization

scholarly journals CancerGD: a resource for identifying and interpreting genetic dependencies in cancer

2016 ◽  
Author(s):  
Stephen Bridgett ◽  
James Campbell ◽  
Christopher J. Lord ◽  
Colm J. Ryan
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Large Scale ◽  
Interaction Networks ◽  
Tumor Cell Lines ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Functional Interaction ◽  
Genetic Aberrations ◽  
Precision Therapeutics

AbstractGenes whose function is selectively essential in the presence of cancer associated genetic aberrations represent promising targets for the development of precision therapeutics. Here we present CancerGD (www.cancergd.org), a resource that integrates genotypic profiling with large-scale loss-of-function genetic screens in tumor cell lines to identify such genetic dependencies. CancerGD provides tools for searching, visualizing, and interpreting these genetic dependencies through the integration of functional interaction networks.

scholarly journals Establishment and Characterization of NCC-DDLPS4-C1: A Novel Patient-Derived Cell Line of Dedifferentiated Liposarcoma

2021 ◽  
Vol 11 (11) ◽  
pp. 1075
Author(s):  
Ryuto Tsuchiya ◽  
Yuki Yoshimatsu ◽  
Rei Noguchi ◽  
Yooksil Sin ◽  
Takuya Ono ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Large Scale ◽  
Dedifferentiated Liposarcoma ◽  
Basic Study ◽  
Therapeutic Drugs ◽  
Candidate Drug ◽  
Deacetylase Inhibitor ◽  
Cell Banks

Dedifferentiated liposarcoma (DDLPS) is a highly malignant sarcoma characterized by the co-amplification of MDM2 and CDK4. Although systemic chemotherapy is recommended for unresectable or metastatic cases, DDLPS is insensitive to conventional chemotherapy, leading to an unfavorable prognosis. Therefore, novel treatment methods are urgently required. Patient-derived cell lines are essential in preclinical studies. Recently, large-scale screening studies using a number of cell lines have been actively conducted for the development of new therapeutic drugs. However, the DDLPS cell line cannot be obtained from public cell banks owing to its rarity, hindering screening studies. As such, novel DDLPS cell lines need to be established. Accordingly, this study aimed to establish a novel DDLPS cell line from surgical specimens. The cell line was named NCC-DDLPS4-C1. NCC-DDLPS4-C1 cells retained copy number alterations corresponding to the original tumors. Further, the cells demonstrated constant growth, spheroid formation, and equivalent invasiveness to MG63 osteosarcoma cells. We also conducted drug screening and integrated the results with those of the previously reported DDLPS cell lines. Consequently, we identified the histone deacetylase inhibitor romidepsin as a novel candidate drug. In conclusion, the NCC-DDLPS4-C1 cell line is a useful tool for the basic study of DDLPS.

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