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Related Experiment Video

Updated: Oct 4, 2025

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models
10:57

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

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Accelerated embryonic stem cell screening with a highly efficient genotyping pipeline.

Roger Caothien1, Charles Yu1, Lucinda Tam1

  • 1Department of Molecular Biology, Genentech, Inc., South San Francisco, CA, USA.

Molecular Biology Reports
|February 2, 2022
PubMed
Summary
This summary is machine-generated.

This study streamlines gene targeting in mouse embryonic stem (ES) cells by integrating magnetic activated cell sorting (MACS) and digital droplet PCR (ddPCR). This novel workflow significantly accelerates the identification of correctly targeted ES cell clones, reducing experimental validation timelines.

Keywords:
ES cellsGene targetingGenotypingMagnetic activated cell sorting MACSMultiplex digital PCR ddPRCTransgenic mice

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Area of Science:

  • Molecular Biology
  • Genetics
  • Stem Cell Biology

Background:

  • Gene targeting in mouse ES cells is crucial for studying gene function.
  • Techniques like conditional alleles and reporter knock-ins are common applications.
  • Accurate identification of targeted clones is essential for experimental success.

Purpose of the Study:

  • To optimize and streamline the screening process for gene targeting in mouse ES cells.
  • To reduce the time between therapeutic target identification and experimental validation.
  • To improve the efficiency and accuracy of identifying correctly targeted ES cell clones.

Main Methods:

  • Integration of magnetic activated cell sorting (MACS) and multiplex droplet digital PCR (ddPCR) into the ES cell screening workflow.
  • Optimization of targeting vector construction and screening procedures.
  • Utilizing adenovirus technology for the removal of drug selection cassettes.

Main Results:

  • Achieved >60% assurance of correctly targeted ES cell clones through optimized MACS and ddPCR integration.
  • Demonstrated a significant reduction in the time required for ES cell screening.
  • Successfully implemented a streamlined workflow for efficient gene targeting.

Conclusions:

  • A novel and effective screening workflow combining ddPCR, multiMACS, and adenovirus recombinase has been developed.
  • This streamlined workflow significantly reduces timelines for gene targeting in mouse ES cells.
  • The improved process accelerates the validation of therapeutic targets.