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Embryonic Stem Cells

Embryonic Stem Cells

Embryonic stem (ES) cells were first discovered in mice in 1981 by Martin Evans. In 1998, James Thomson identified a method to isolate embryonic stem cells from humans. Human embryonic stem cells (hESCs) are obtained from 3-5 day old embryos that remain unused after an in vitro fertilization procedure.
ES cells are grown in a culture medium where they can divide indefinitely, creating ES cell lines. Under certain conditions, ES cells can differentiate, either spontaneously into a variety of...

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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Updated: Apr 20, 2026

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

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

Published on: February 2, 2016

Genome editing in human stem cells.

Susan M Byrne¹, Prashant Mali¹, George M Church¹

¹Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

Methods in Enzymology

|November 16, 2014

Summary

This summary is machine-generated.

This study presents an efficient protocol for genetic engineering in human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 technology. The method achieves high gene targeting efficiency without selection, making it accessible for most laboratories.

Keywords:

CRISPR Cas9 nuclease Gene targeting Human genome engineering Induced pluripotent stem cells Transfection

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

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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Published on: September 25, 2019

Area of Science:

Molecular Biology
Genetics
Stem Cell Biology

Background:

Custom-engineered nucleases like CRISPR/Cas9, ZFN, and TALEN enable precise genetic modifications in human cells.
Engineered DNA breaks can disrupt genes or facilitate gene insertions and point mutations via donor vectors.
Engineering human embryonic stem cells and iPSCs presents challenges due to transfection difficulty and DNA damage sensitivity.

Purpose of the Study:

To describe a straightforward protocol for genetic engineering in human induced pluripotent stem cells (iPSCs).
To achieve high gene targeting efficiency in iPSCs without the need for subsequent selection steps.
To provide guidance on designing optimal sgRNA target sites and donor vectors for gene editing.

Main Methods:

Utilizing transient transfection of plasmids and/or single-stranded oligonucleotides for gene editing.
Employing custom-engineered sequence-specific nucleases, including CRISPR/Cas9.
Developing strategies for identifying, cloning, and genotyping successfully edited cells.

Main Results:

Achieved typical gene targeting efficiencies between 1% and 10% in human iPSCs.
Demonstrated a protocol that does not require post-editing selection steps.
Provided practical methods for designing and validating gene editing components.

Conclusions:

The described protocol offers an accessible and efficient method for genetic engineering in human iPSCs.
The findings facilitate precise genetic modifications in stem cells for research and therapeutic applications.
Alternative gene editing strategies, such as viral delivery and modified Cas9 systems, were also discussed.