Gene Targeting Techniques
Gene Targeting Techniques - 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...
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.
07:27
CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira
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2025
This video demonstrates a CRISPR interference-based gene silencing in pathogenic Leptospira. Targeted repression of gene expression using a CRISPR/Cas system that blocks mRNA formation helps identify candidate genes for pathological and pharmacological studies.
11:29
Technique to Target Microinjection to the Developing Xenopus Kidney
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Cited by 17 •
2016
Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.
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.
FAQs
Two main targeting strategies provide locus specificity in different ways. Sequence homology uses matching DNA regions to direct the modification, whereas programmable nucleases recognize a selected genomic locus and introduce a cut. The design determines whether a donor template accompanies the targeting strategy, helping focus the experiment on one defined genetic site.
Cellular DNA-repair pathways convert the targeting event into the final genetic outcome. After a cut or donor-template-directed event, repair can produce a mutation, deletion, insertion, or replacement at the selected locus. This step matters because the resulting DNA change, rather than the targeting signal alone, determines which genetic alteration researchers can analyze.
A defined location connects the engineered change to a particular gene or regulatory region, making the resulting phenotype easier to associate with that genetic alteration. This precision supports experiments asking whether a specific sequence contributes to gene function or regulation, rather than treating an unlocalized change as the experimental cause.
A knockout study is designed to disrupt or remove gene activity through a targeted mutation or deletion, whereas a knock-in study introduces a planned sequence at the locus. This distinction lets researchers compare outcomes from removing genetic information with outcomes from adding or replacing it in cells or organisms.
Researchers first select the genomic locus and design a targeting strategy based on sequence homology or a programmable nuclease. They then introduce the relevant cut, donor template, or both, and rely on cellular DNA-repair pathways to generate the intended mutation, insertion, deletion, or replacement. The resulting genetic change can then be examined for its biological effect.
They are useful when researchers need to connect a defined genetic change with gene function, regulation, or an observable phenotype. Applications include functional genomics, disease modeling, and development of engineered cells and organisms. These uses extend from testing the role of individual sequences to building biological systems with deliberately altered genetic information.
By placing a designed mutation or sequence change at a chosen locus, researchers can create a genetic context for studying how that alteration affects biology. Disease modeling uses this relationship to investigate relevant genetic changes, while phenotype analysis helps determine whether the engineered modification produces an observable result linked to gene function or regulation.