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Related Concept Videos

In-vitro Mutagenesis01:16

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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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Updated: Apr 6, 2026

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models
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Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells.

Matyas Flemr1, Marc Bühler1

  • 1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Petersplatz 10, 4003 Basel, Switzerland.

Cell Reports
|July 21, 2015
PubMed
Summary
This summary is machine-generated.

We developed a reporter system to improve homologous recombination (HR) efficiency for complex genome engineering. This method enables highly efficient, simultaneous DNA integration at two sites in mouse embryonic stem cells (mESCs).

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Area of Science:

  • Molecular Biology
  • Genome Engineering
  • Stem Cell Biology

Background:

  • Engineered nucleases like CRISPR/Cas9 and TALENs induce DNA double-strand breaks (DSBs) to facilitate exogenous DNA knockin via homologous recombination (HR).
  • Current HR efficiencies limit complex genomic modifications, such as simultaneous integration at multiple sites.

Purpose of the Study:

  • To develop a novel reporter system for enriching cells with engineered nuclease-assisted HR events.
  • To enhance the efficiency of complex, multi-site genome modifications in mouse embryonic stem cells (mESCs).

Main Methods:

  • Development of a reporter system to select for successful HR events.
  • Application of the reporter system in mESCs using engineered nucleases.
  • Validation of seamless, biallelic integration of DNA fragments.

Main Results:

  • Achieved single-step, biallelic, and seamless integration of two loxP sites for inducible gene knockout.
  • Demonstrated high-efficiency biallelic endogenous gene tagging.
  • Significantly reduced time and resources for generating conditional knockout mESCs.

Conclusions:

  • The developed reporter system greatly enhances the efficiency of nuclease-assisted homologous recombination.
  • This approach facilitates complex genomic modifications, including simultaneous dual-site integration.
  • The method streamlines the generation of conditional knockout mESCs, saving time and resources.