scholarly journals One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines

2021 ◽  
pp. mbc.E21-02-0051
Author(s):  
Tsz Kwan Yeung ◽  
Ho Wai Lau ◽  
Hoi Tang Ma ◽  
Randy Y.C. Poon
Keyword(s):  
Gene Silencing ◽  
Cell Lines ◽  
Human Cell ◽  
Mammalian Cells ◽  
Essential Genes ◽  
Gene Inactivation ◽  
Human Cell Lines ◽  
Efficient Generation ◽  
Multiple Genes ◽  
One Step

Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines

2018 ◽  
Vol 293 (3) ◽  
pp. 769-783 ◽  
Author(s):  
Fei Wang ◽  
Tao Guo ◽  
Hongmei Jiang ◽  
Ruobi Li ◽  
Ting Wang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines ◽  
Aldh2 Gene

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.

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

VNB-mediated endosomal escape triggers robust gene silencing in human cell lines

2020 ◽  
Author(s):  
Juan Fraire ◽  
Gaëlle Houthaeve ◽  
Jing Liu ◽  
Laurens Raes ◽  
Stephan Stremersch ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Cell Lines ◽  
Human Cell ◽  
Endosomal Escape ◽  
Human Cell Lines

scholarly journals Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines.

1986 ◽  
Vol 103 (4) ◽  
pp. 1595-1603 ◽  
Author(s):  
P J Brown ◽  
R L Juliano
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Lines ◽  
Human Cell ◽  
Mammalian Cells ◽  
Surface Glycoprotein ◽  
Cell Surface Receptor ◽  
Human Cell Lines ◽  
Cell Surface Glycoprotein ◽  
Fibronectin Receptor

We have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. We now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bands are closely related polypeptides.

scholarly journals Genotypic Stability, Segregation and Selection in Heteroplasmic Human Cell Lines Containing np 3243 Mutant mtDNA

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 363-380
Author(s):  
Sanna K Lehtinen ◽  
Nicole Hance ◽  
Abdellatif El Meziane ◽  
M Katariina Juhola ◽  
K Martti I Juhola ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Mammalian Cells ◽  
Cell Clone ◽  
Rare Event ◽  
Chromosome 9 ◽  
Human Cell Lines ◽  
Mtdna Copy Number ◽  
Short Period ◽  
Mitochondrial Genotype

Abstract The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (≥1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (<8 wk), indicating selection. One such episode was associated with a gain of chromosome 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were generally reestablished within a few weeks, in a reproducible but clone-specific fashion. This occurred independently of any straightforward phenotypic selection at the individual cell-clone level. Our findings are consistent with several alternate views of mtDNA organization in mammalian cells. One model that is supported by our data is that mtDNA is found in nucleoids containing many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids, under nuclear genetic control. Abrupt remodeling of nucleoids in vivo would have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression.

Investigations of the influence of microgravity on the state of human cell lines

2004 ◽  
Vol 10 (5-6) ◽  
pp. 226-228
Author(s):  
L.M. Nosach ◽  
◽  
O.Yu. Povnitsa ◽  
V.L. Zhovnovata ◽  
◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
The State ◽  
Human Cell Lines
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