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Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

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CRISPR/Cas9-Mediated Modification of PTP Expression.

Carolin Lossius¹, Anne Kresinsky^1,2, Laura Quiet¹

¹Institute for Molecular Cell Biology, CMB - Center for Molecular Biomedicine; University Hospital Jena, Jena, Germany.

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

|December 26, 2023

Summary

Researchers developed CRISPR/Cas9 genome editing methods to inactivate or activate protein tyrosine phosphatase (PTP) gene expression. These techniques are broadly applicable for studying PTP functions in mammalian cells.

Keywords:

CRISPR Cas9 Flow cytometry Gene expression Immune detection Nonhomologous end joining Protein tyrosine phosphatase Synergistic activation mediators

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Area of Science:

Molecular Biology
Genetics
Cell Biology

Background:

Protein tyrosine phosphatases (PTPs) are crucial regulators of cell signaling.
Analyzing PTP function often involves altering their gene expression in mammalian cells.

Purpose of the Study:

To establish CRISPR/Cas9 genome editing methods for PTP gene manipulation.
To demonstrate these methods for PTPRJ (Dep-1, CD148) and PPTRC (CD45) inactivation and activation.

Main Methods:

CRISPR/Cas9 genome editing was employed for gene inactivation.
CRISPR/Cas9 was utilized for transcriptional activation of PTP genes.
Methods were validated using specific PTPs like PTPRJ and PPTRC.

Main Results:

Successful inactivation of PTPRJ and PPTRC genes was achieved using CRISPR/Cas9.
Transcriptional activation of PTPRJ and PPTRC was successfully demonstrated.
The developed genome editing approaches are effective for PTP gene modulation.

Conclusions:

CRISPR/Cas9 provides a powerful tool for functional analysis of PTPs.
The presented methods offer a versatile approach for studying various PTPs.
This work facilitates deeper understanding of PTP roles in cell physiology.