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Related Experiment Video

Updated: May 30, 2026

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Published on: May 10, 2020

Gene targeting in human pluripotent stem cells.

Ying Liu¹, Mahendra Rao

¹Department of Reproductive Medicine, UCSD Medical Center, University of California, San Diego, San Diego, CA, USA. yil032@ucsd.edu

Methods in Molecular Biology (Clifton, N.J.)

|August 9, 2011

Summary

We developed an efficient gene targeting protocol for human pluripotent stem cells (hPSCs) using homologous recombination (HR). This method overcomes previous efficiency limitations, enabling precise genetic modifications for disease modeling and reporter line generation in hPSCs.

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Last Updated: May 30, 2026

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

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Published on: May 10, 2020

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

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Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Published on: August 23, 2014

Area of Science:

Stem cell biology
Molecular genetics
Human pluripotent stem cells (hPSCs)

Background:

Targeted homologous recombination (HR) is crucial for gene function studies in mouse embryonic stem cells (mESCs).
Low efficiency of gene targeting has limited its application in human pluripotent stem cells (hPSCs).

Purpose of the Study:

To establish an efficient protocol for gene targeting in hPSCs.
To overcome the technical challenges associated with low targeting efficiency in human cell lines.

Main Methods:

Electroporation-based delivery of targeting vectors into hPSCs.
Detailed procedures for cell preparation, antibiotic selection, and marker gene excision.
Application of homologous recombination for precise genomic modifications.

Main Results:

Successfully established an efficient gene targeting protocol for hPSCs.
Demonstrated the feasibility of single-allele targeting for various applications.
Protocol allows for generating knock-in reporter lines, disease models, and genetic repair.

Conclusions:

The developed protocol significantly enhances gene targeting efficiency in hPSCs.
This method facilitates diverse applications including disease modeling and genetic manipulation in hPSCs.
Broad applicability for generating lineage-specific reporters and correcting mutations in hPSC-based disease models.