Targeted Gene Disruption
Targeted Gene Disruption - Related Videos
08:56
Recombineering and Gene Targeting
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2023
One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based...
12:06
Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning
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Cited by 1 •
2017
We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.
08:43
Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics
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Cited by 9 •
2012
Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.
08:48
Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
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Cited by 5 •
2017
A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.
05:33
Biolistic-Mediated Gene Transfer: A Technique to Deliver Gene of Interest in Target Cells via a Biolistic Gene Gun
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2025
In this video, we demonstrate the protocol of biolistic gene gun-mediated gene delivery in cultured cells. This method provides a simple method of stable genetic transformation using a minimal quantity of DNA.
FAQs
The guide RNA provides sequence recognition by matching a selected DNA region, positioning the Cas9 nuclease at that site. Cas9 then makes a double-strand break rather than altering DNA indiscriminately. This targeting step is central to assigning the resulting genetic change to one chosen gene, allowing researchers to examine how disruption affects the cell or organism.
They use different repair routes after the Cas9-induced break. Nonhomologous end joining is error-prone and can create insertions or deletions that disable the selected gene. In contrast, providing a donor template can support precise replacement of the intended sequence. The choice therefore determines whether the experiment primarily seeks gene inactivation or a defined sequence change.
The repair outcome determines what genetic change is actually being studied. Error-prone repair may disable the selected gene through insertions or deletions, whereas donor-template repair may produce a precise replacement. Interpreting the resulting consequences therefore requires distinguishing loss of gene activity from a defined sequence change, especially when connecting DNA alterations with traits or disease-related mechanisms.
A typical workflow begins by choosing the gene and matching DNA sequence, then using a guide RNA to direct Cas9 to that location. After the nuclease creates a double-strand break, the cell repairs the DNA through nonhomologous end joining or with a donor template. The resulting inactivation or precise replacement can then be related to observed consequences.
Researchers apply it when they need to connect a selected gene with a biological trait or consequence. In genetics, the approach supports studies of gene function in cells or organisms, modeling inherited disorders, validating disease mechanisms, and evaluating potential therapeutic targets. These uses make it a link between deliberate DNA alteration and experimentally observed biological outcomes.
In disease research, researchers can use the resulting gene inactivation or precise replacement to test whether a gene contributes to an inherited disorder or disease mechanism. If the altered cell or organism shows relevant consequences, the gene becomes more strongly connected to that process, while the same framework can help assess whether it is a potential therapeutic target.