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In-vitro Mutagenesis

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: Nov 6, 2025

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Published on: August 24, 2022

Precise BAC targeting of genetically polymorphic mouse ES cells.

Tahsin Stefan Barakat¹, Eveline Rentmeester, Frank Sleutels

¹Department of Reproduction and Development, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.

Nucleic Acids Research

|July 9, 2011

Summary

This summary is machine-generated.

This study introduces a novel bacterial artificial chromosome (BAC) targeting method for precise gene editing in mouse embryonic stem (ES) cells. The strategy utilizes restriction fragment length polymorphisms (RFLPs) for efficient knockout allele generation and reliable detection.

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Last Updated: Nov 6, 2025

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

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Published on: August 24, 2022

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

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Published on: November 20, 2018

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Published on: April 27, 2017

Area of Science:

Genetics and Genomics
Molecular Biology
Stem Cell Biology

Background:

Bacterial artificial chromosomes (BACs) facilitate high homologous recombination efficiency in embryonic stem (ES) cells due to long sequence homology.
Accurate identification of targeted ES cell clones is crucial for genetic manipulation.

Purpose of the Study:

To introduce a novel BAC targeting method for efficient and precise gene knockout in mouse ES cells.
To utilize restriction fragment length polymorphisms (RFLPs) for identifying correctly targeted ES cell clones.

Main Methods:

Employing BAC targeting in polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines.
Generating knockout alleles by targeting RFLPs within open reading frames or introducing transcription stop cassettes.
Utilizing allele-specific PCR for identification of Rnf12 heterozygous knockout ES cells from genomic DNA or cDNA.

Main Results:

Demonstrated successful generation of knockout alleles by disrupting RFLPs or using transcription stop cassettes.
Successfully generated Rnf12 heterozygous knockout ES cells.
Validated the efficiency and precision of the BAC targeting strategy with PCR-based readout.

Conclusions:

The novel BAC targeting method offers high efficiency and precision for gene editing in ES cells.
The strategy provides a convenient and reliable PCR-based readout for detecting correct targeting events.
This approach facilitates the generation of specific knockout alleles for genetic studies.