Gene Deletion
Gene Deletion - Related Videos
14:07
Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
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Cited by 9 •
2012
An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.
13:06
The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions
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Cited by 13 •
2012
The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.
02:27
Generating Double-Crossover Recombinants of Pseudomonas aeruginosa for Targeted Gene Deletion
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2025
Source: Valentine, M. E., et al. Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection. J. Vis. Exp. (2020).This video demonstrates the generation of double-crossover recombinants in Pseudomonas aeruginosa using a plasmid-based gene deletion strategy. The process involves selecting bacteria that harbor a non-replicative plasmid integrated into the genome via a single crossover and then inducing a second...
11:24
Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs
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Cited by 8 •
2017
Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.
03:36
Generating Gene Deletions in E. coli via Phage-Mediated Transfer of an Excisable Antibiotic Cassette
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2026
Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp. (2018)This video demonstrates the phage-mediated transfer of donor DNA carrying an excisable antibiotic resistance cassette into Escherichia coli. The integrated cassette replaces the target gene via homologous recombination, resulting in a stable gene deletion.
FAQs
Homologous recombination and CRISPR-Cas can both be directed toward a selected genomic site, but they provide different routes for changing it. Homologous recombination supports replacement using a designed DNA template, whereas CRISPR-Cas directs a DNA cut and can be followed by repair that joins the resulting ends or incorporates that template. The choice therefore affects how the intended alteration is produced.
Cellular DNA repair determines the final result after the targeted change is initiated. Repair may join the DNA ends, or it may incorporate a designed template, so the resulting sequence depends on which repair outcome occurs. This step matters because the experiment is interpreted through whether the altered cells or organisms show the expected loss of normal gene-product function.
Comparing mutant cells or organisms with unmodified controls is essential for linking an observed phenotype to the deletion. Differences in development, physiology, or disease-related pathways become informative when the unmodified group provides the comparison point. This design turns a sequence-level alteration into evidence about the gene's contribution to a biological process.
An experiment begins by selecting the genomic sequence to alter. Researchers then create the change through homologous recombination or CRISPR-Cas, allowing cellular repair to join DNA ends or use a designed template. Finally, they compare altered cells or organisms with unmodified controls and assess effects on development, physiology, or disease-related pathways.
Researchers can assess effects across several biological levels, including development, physiology, and disease-related pathways. The key interpretation comes from comparing altered cells or organisms with unmodified controls, then determining which outcomes differ between them. This makes the model useful for connecting a gene's disruption with observable biological changes rather than examining the DNA sequence alone.
Functional genomics uses these models to connect a gene disruption with measurable biological consequences, helping researchers assign function and analyze pathways. The same evidence can be considered in disease-related contexts: if altering a gene changes a relevant pathway, researchers can evaluate that gene as a potential therapeutic target. This makes deletion models useful for both biological discovery and target assessment.