CRISPR‐Cas9‐mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes

Oct 12, 2019Journal of cellular physiology

Removing the Prkdc gene in mouse stem cells changes the activity of genes that keep cells able to become any cell type

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Abstract

The bi-allelic Prkdc-knockout cell line showed overexpression of pluripotency-associated genes, including Nanog and Sox-2.

Prkdc encodes a key protein involved in DNA repair and other critical biological functions.
The knockout of Prkdc in mouse embryonic stem cells resulted in typical stem cell morphology and cell cycle distribution.
Alkaline phosphatase activity was maintained in the Prkdc-knockout cell line.
Expression of several germ layer markers was altered in Prkdc-knockout lines.
These findings suggest that Prkdc may influence pluripotency and differentiation in embryonic stem cells.

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Prkdc encodes for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) playing a key role in nonhomologous end joining pathway during DNA double-strand break repair and also influencing the homologous recombination (HR) repair system by phosphorylation of proteins involved in HR. In addition, Prkdc has other critical functions in biological processes, such as transcriptional regulation, telomere stability, apoptosis, and metabolism. DNA-PKcs upregulates during in vitro differentiation of mouse embryonic stem cells (mESCs). To address the potential role of Prkdc in mESCs pluripotency and in vitro differentiation into ectoderm, mesoderm, and endoderm germ layers under normal physiological conditions, a bi-allelic Prkdc-knockout cell line was generated in the present study by employing CRISPR/Cas9 system, and subsequently, its potential role in stemness and development was studied. The results of the study showed that the expression of pluripotency-associated genes, including Nanog and Sox-2 were overexpressed in the bi-allelic Prkdc-knockout cell line. Also, bi-allelic Prkdc-knockout cell line was shown to have typical mESCs cell morphology, cell cycle distribution, and alkaline phosphatase activity. Furthermore, the results of the study revealed that the expression of several germ layer markers is modulated in Prkdc-knockout lines. In conclusion, the findings of our study demonstrated the role of Prkdc during differentiation and development of ESCs.

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CRISPR‐Cas9‐mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes

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