Knockout Mutant Screening
Knockout Mutant Screening - Related Videos
08:25
Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
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Cited by 6 •
2017
The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.
12:25
Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells
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2026
Here, we present a protocol for generating chemoresistant cells using a dose-escalation approach, followed by a CRISPR/Cas9-based screen using a focused sgRNA library targeting epigenetic modifiers to identify regulators of acquired chemoresistance. The protocol also provides multiple optimization points tailored to chemoresistant cell models, offering a robust framework for researchers investigating resistance mechanisms.
14:56
A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants
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Cited by 18 •
2012
Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.
10:50
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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Cited by 10 •
2016
We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.
11:44
Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants
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Cited by 28 •
2015
Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.
FAQs
The key comparison is between a mutant and an unmodified control. When loss of a gene produces a reproducible developmental difference under the same defined conditions, that contrast provides evidence that the gene contributes to the affected process. Examining morphology, timing, cell behavior, or viability helps specify how its absence changes development.
Morphology can reveal altered body or tissue formation, while developmental timing can show when a process is delayed or changed. Cell behavior and viability provide additional perspectives on how development is affected. Using several measurable traits rather than a single observation can distinguish different kinds of phenotypic change in the developing organism.
Systematic screening can identify patterns in how different gene disruptions affect development, including interactions among genes. Such findings extend analysis beyond an individual phenotype by indicating that gene functions may be connected within developmental processes. The resulting candidates can then be selected for deeper mechanistic study of patterning, tissue formation, or signaling.
An unmodified control establishes the developmental comparison needed to associate an observed difference with gene disruption. Defined conditions make that comparison more consistent by limiting variation in the experimental setting. Together, they help researchers judge whether changes in morphology, timing, cell behavior, or viability are associated with the knockout rather than an uncontrolled difference.
Researchers first create or collect mutants in which a target gene is disrupted. They then examine mutant development alongside an unmodified control under defined conditions and record measurable differences. Observations may include morphology, developmental timing, cell behavior, or viability, after which informative phenotypes guide selection of genes for further study.
This approach is particularly useful when researchers want to connect specific genes with embryonic patterning, tissue formation, or developmental signaling. Comparing developmental outcomes after gene disruption can identify genes associated with these processes and reveal phenotypes that merit closer investigation. The strategy therefore supports systematic discovery as well as focused analysis of individual developmental genes.
A phenotype provides a starting point for interpreting the disrupted gene's possible developmental function. Researchers can organize informative changes by traits such as morphology, timing, cell behavior, or viability, and use systematic results to identify interactions among genes. Promising findings then supply candidates for deeper mechanistic study rather than serving as a final explanation by themselves.