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Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Related Experiment Video

Updated: Jun 15, 2026

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
09:40

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Published on: January 3, 2015

DelsGate: a robust and rapid method for gene deletion.

María D García-Pedrajas1, Marina Nadal, Timothy Denny

  • 1Estación Experimental "La Mayora", CSIC, Algarrobo-Costa, Málaga, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|March 19, 2010
PubMed
Summary
This summary is machine-generated.

We developed DelsGate, a fast and simple method for creating gene deletion mutants. This technique combines PCR and Gateway cloning to generate precise deletion constructs in just two days, aiding high-throughput gene function studies.

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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
09:40

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Gene deletion is crucial for understanding gene function, especially with the increasing number of identified genes in the genomics era.
  • There is a high demand for efficient, high-throughput methods for generating gene deletion mutants.
  • Existing methods can be time-consuming and complex, necessitating streamlined approaches.

Purpose of the Study:

  • To develop a fast, simple, and universal method for generating gene deletion constructs.
  • To streamline the process of creating deletion mutants for high-throughput functional genomics.
  • To adapt existing molecular biology tools for efficient gene deletion construction.

Main Methods:

  • Developed DelsGate (Deletion via Gateway), a method combining PCR, Gateway cloning, and I-SceI endonuclease.
  • Utilized PCR to amplify 5' and 3' gene flanks (1 kb each).
  • Performed in vitro Gateway cloning followed by in vivo recombination in Escherichia coli to generate precise deletion constructs.

Main Results:

  • DelsGate enables the generation of precise deletion constructs in a universal and robust manner within 2 days.
  • Modified Gateway cloning vectors include selectable markers for Ascomycetes and Ustilago maydis transformation.
  • Successfully applied DelsGate for gene deletion in the fungus Ustilago maydis and for creating unmarked deletions in Ralstonia solanacearum.

Conclusions:

  • DelsGate significantly speeds up the generation of deletion mutants, facilitating high-throughput gene function studies.
  • The method is adaptable for various fungal species and other genetic manipulations, including unmarked deletions in bacteria.
  • DelsGate offers a robust and efficient platform for genetic research in diverse microorganisms.