scholarly journals Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens

2017 ◽  
Author(s):  
Traver Hart ◽  
Amy Tong ◽  
Katie Chan ◽  
Jolanda Van Leeuwen ◽  
Ashwin Seetharaman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Viral Vectors ◽  
Human Cancer ◽  
Gene Knockout ◽  
Essential Genes ◽  
Human Cell Lines ◽  
Protein Coding ◽  
Mammalian Cell Lines ◽  
Genome Scale

AbstractThe adaptation of CRISPR/Cas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs), targeting human protein-coding genes and encoded in viral vectors, have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries, using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, to refine and expand our previously defined set of human core essential genes, from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2), plus empirical data from six CRISPR knockout screens, to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

Cytoplasmic transfer of a determinant for chloramphenicol resistance between mammalian cell lines

1978 ◽  
Vol 201 (1142) ◽  
pp. 73-85 ◽  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Mitochondrial Protein ◽  
Human Cell Lines ◽  
Resistant Variant ◽  
Chloramphenicol Resistance ◽  
Resistance Phenotype ◽  
Whole Cells ◽  
Mammalian Cell Lines ◽  
Cell Line Hela

A genetic analysis of the resistance phenotype of a recently described chloramphenicol-resistant variant derived from the human cell line, HeLa (MC63), has been undertaken. Whole cells or enucleated fragments, produced by treatment with cytochalasin B, were fused with chloram­phenicol-sensitive mouse, or human cells. Enucleated cells (cytoplasts) act as very efficient donors of the resistance phenotype in fusions with other human cell lines derived from HeLa. We conclude that chloram­phenicol resistance is determined cytoplasmically. Transfer of resistance to unrelated human cell lines occurred at much lower frequency and we were unable to demonstrate transfer to mouse cells. An examination of mitochondrial protein synthesis in the fusion products of cytoplasts and whole cells suggested that mixed populations of mitochondria from both parental cells were maintained under the conditions of selection.

scholarly journals MetaTropismDB: a database of organ-specific metastasis induced by human cancer cell lines in mouse models

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Matteo Giulietti ◽  
Marco Bastianoni ◽  
Monia Cecati ◽  
Annamaria Ruzzo ◽  
Massimo Bracci ◽  
...  
Keyword(s):  
Mouse Models ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Cell ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
Human Cell Lines ◽  
Human Cancer Cell ◽  
Human Cancer Cell Lines ◽  
Organ Specific

Abstract The organotropism is the propensity of metastatic cancer cells to colonize preferably certain distant organs, resulting in a non-random distribution of metastases. In order to shed light on this behaviour, several studies were performed by the injection of human cancer cell lines into immunocompromised mouse models. However, the information about these experiments is spread in the literature. For each xenograft experiment reported in the literature, we annotated both the experimental conditions and outcomes, including details on inoculated human cell lines, mouse models, injection methods, sites of metastasis, organs not colonized, rate of metastasis, latency time, overall survival and the involved genes. We created MetaTropismDB, a freely available database collecting hand-curated data useful to highlight the mechanisms of organ-specific metastasis. Currently, it stores the results of 513 experiments in which injections of 219 human cell lines have been carried out in mouse models. Notably, 296 genes involved in organotropic metastases have been collected. This specialized database allows the researchers to compare the current results about organotropism and plan future experiments in order to identify which tumour molecular signatures establish if and where the metastasis will develop. Database URL:  http://www.introni.it/Metastasis/metastasis.html

scholarly journals Gene knockout shows that PML (TRIM19) does not restrict the early stages of HIV-1 infection in human cell lines

2017 ◽  
Author(s):  
Nasser Masroori ◽  
Pearl Cherry ◽  
Natacha Merindol ◽  
Jia-xin Li ◽  
Caroline Dufour ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Gene Knockout ◽  
Human Cells ◽  
Restriction Factor ◽  
Type I ◽  
Human Cell Lines ◽  
Early Stages ◽  
Human T Cell ◽  
Hiv 1

AbstractThe PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. By contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNAi with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used CRISPR-Cas9 to knock out its gene in epithelial, lymphoid and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Over-expression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5α was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages.ImportancePML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide unambiguous, knockout-based evidence that PML does not restrict the early post-entry stages of HIV-1 infection in a variety of human cell types and does not participate in the inhibition of HIV-1 by IFN-I. Although this study does not exclude the possibility of other mechanisms by which PML may interfere with HIV-1, we nonetheless demonstrate that PML does not generally act as an HIV-1 restriction factor in human cells and that its presence is not required for IFN-I to stimulate the expression of anti-HIV-1 genes. These results contribute to uncovering the landscape of HIV-1 inhibition by ISGs in human cells.

scholarly journals Gene Knockout Shows That PML (TRIM19) Does Not Restrict the Early Stages of HIV-1 Infection in Human Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Nasser Masroori ◽  
Pearl Cherry ◽  
Natacha Merindol ◽  
Jia-xin Li ◽  
Caroline Dufour ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Gene Knockout ◽  
Human Cells ◽  
Restriction Factor ◽  
Type I ◽  
Human Cell Lines ◽  
Early Stages ◽  
Human T Cell ◽  
Hiv 1

ABSTRACTThe PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. In contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNA interference, with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used the clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9) system to knock out its gene in epithelial, lymphoid, and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Overexpression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5α (another human TRIM protein), and TRIM5α was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages.IMPORTANCEPML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide unambiguous, knockout-based evidence that PML does not restrict the early postentry stages of HIV-1 infection in a variety of human cell types and does not participate in the inhibition of HIV-1 by IFN-I. Although this study does not exclude the possibility of other mechanisms by which PML may interfere with HIV-1, we nonetheless demonstrate that PML does not generally act as an HIV-1 restriction factor in human cells and that its presence is not required for IFN-I to stimulate the expression of anti-HIV-1 genes. These results contribute to uncovering the landscape of HIV-1 inhibition by ISGs in human cells.

scholarly journals A gene signature for gold nanoparticle-exposed human cell lines

2015 ◽  
Vol 4 (2) ◽  
pp. 365-375 ◽  
Author(s):  
Ruei-Yue Liang ◽  
Hsin-Fang Tu ◽  
Xiaotong Tan ◽  
Yu-Shan Yeh ◽  
Pin Ju Chueh ◽  
...  
Keyword(s):  
Gold Nanoparticle ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Cdna Microarray ◽  
Human Cell ◽  
Gene Signature ◽  
Human Cell Lines ◽  
Biological Assays ◽  
Mammalian Cell Lines

A unique four-gene signature for AuNP exposure was identified using the cDNA microarray and evaluated by qPCR and biological assays in mammalian cell lines.

scholarly journals Investigating the Selectivity of Novel Chalcones for Human Cancer Cells Compared to Non‐Cancerous Human Cell Lines

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Christopher R.T. Stang ◽  
Laura R. Inbody ◽  
Mikaela C. Hickey ◽  
Ahmed H. Mohamed ◽  
Courtney A. Hollenbacher ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cancer ◽  
Human Cell Lines ◽  
Human Cancer Cells

scholarly journals HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors

2021 ◽  
Vol 9 ◽  
Author(s):  
Evan Tan ◽  
Cara Sze Hui Chin ◽  
Zhi Feng Sherman Lim ◽  
Say Kong Ng
Keyword(s):  
Host Cell ◽  
Cell Lines ◽  
Recombinant Proteins ◽  
Human Cell ◽  
Viral Vectors ◽  
Animal Cell ◽  
Hek293 Cells ◽  
Human Cell Lines ◽  
Post Translational Modifications ◽  
Serum Free Suspension

Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more “human-like” post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.

scholarly journals An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines

2020 ◽  
Author(s):  
Marina Wright Muelas ◽  
Ivayla Roberts ◽  
Farah Mughal ◽  
Steve O’Hagan ◽  
Philip J. Day ◽  
...  
Keyword(s):  
Blood Serum ◽  
Cell Lines ◽  
Human Cell ◽  
Time Course ◽  
Phospholipid Bilayer ◽  
Untargeted Metabolomics ◽  
Human Cell Lines ◽  
Biologically Relevant ◽  
Mammalian Cell Lines ◽  
Transporter Expression

AbstractIntroductionIt is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites.ObjectivesBlood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to measure them.MethodsWe develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the ‘exometabolome’ or metabolic footprint).ResultsOur method measures some 4,000-5,000 metabolic features in both ES+ and ES− modes. We show that the metabolic footprints of different cell lines differ greatly from each other.ConclusionOur new, 15-minute untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum, and its relation to differences in transporter expression.

CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines

2021 ◽  
pp. 29-42
Author(s):  
Kimberly S. Huggler ◽  
Nicholas J. Rossiter ◽  
Kyle M. Flickinger ◽  
Jason R. Cantor
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Essential Genes ◽  
Human Cell Lines

scholarly journals Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

2018 ◽  
Author(s):  
Isac Lee ◽  
Roham Razaghi ◽  
Timothy Gilpatrick ◽  
Michael Molnar ◽  
Norah Sadowski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Large Scale ◽  
Human Cancer ◽  
Chromatin Accessibility ◽  
Nanopore Sequencing ◽  
Human Cell Lines ◽  
Sequencing Data ◽  
Structural Variations ◽  
Genetic Changes

ABSTRACTUnderstanding how the genome and the epigenome work together to control gene transcription has applications in our understanding of diseases such as human cancer. In this study, we combine the ability of NOMe-seq to simultaneously evaluate CpG methylation and chromatin accessibility, with long-read nanopore sequencing technology, a method we call nanoNOMe. We generated >60Gb whole-genome nanopore sequencing data for each of four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231) including repetitive regions inaccessible by short read sequencing. Using the long reads, we find that we can observe phased methylation and chromatin accessibility, large scale pattern changes, and genetic changes such as structural variations from a single assay.

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