Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting: SimpleCells with Homogeneous GalNAc O-glycosylation Allow Isolation of the O-glycoproteome by One-Step Lectin Affinity Chromatography

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

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Zinc-Finger Nucleases-Based Genome Engineering to Generate Isogenic Human Cell Lines

Methods in Molecular Biology - Synthetic Gene Networks ◽

10.1007/978-1-61779-412-4_8 ◽

2011 ◽

pp. 145-156 ◽

Cited By ~ 6

Author(s):

Anne-Kathrin Dreyer ◽

Toni Cathomen

Keyword(s):

Cell Lines ◽

Zinc Finger ◽

Human Cell ◽

Genome Engineering ◽

Zinc Finger Nucleases ◽

Human Cell Lines

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Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

Nucleic Acids Research ◽

10.1093/nar/gkv1338 ◽

2015 ◽

Vol 44 (6) ◽

pp. e54-e54 ◽

Cited By ~ 8

Author(s):

Sivasundaram Karnan ◽

Akinobu Ota ◽

Yuko Konishi ◽

Md Wahiduzzaman ◽

Yoshitaka Hosokawa ◽

...

Keyword(s):

Gene Targeting ◽

Cell Lines ◽

Human Cell ◽

Human Cell Lines ◽

2A Peptide ◽

Improved Methods

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A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance—GFP fusion gene

BioTechniques ◽

10.2144/0000113911 ◽

2012 ◽

Vol 53 (3) ◽

pp. 141-152 ◽

Cited By ~ 2

Author(s):

Yuko Konishi ◽

Sivasundaram Karnan ◽

Miyuki Takahashi ◽

Akinobu Ota ◽

Lkhagvasuren Damdindorj ◽

...

Keyword(s):

Antibiotic Resistance ◽

Gene Targeting ◽

Cell Lines ◽

Human Cell ◽

Fusion Gene ◽

Human Cell Lines ◽

Targeting Efficiency

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

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0051 ◽

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.

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