Knock out aryl hydrocarbon receptor gene in adipose-derived mesenchymal stem cell line (PT-5006) by CRISPR/Cas9

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Authors

  • Le Hoang Duc \(^1\) Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam
    \(^2\) Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam
    https://orcid.org/0000-0001-9780-1282
  • Vu Thi Tien \(^1\) Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam
  • Nguyen Trung Nam \(^1\) Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam
    \(^2\) Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam
    https://orcid.org/0000-0003-2369-954X

DOI:

https://doi.org/10.15625/vjbt-20645

Keywords:

Adipose-derived MSCs, aryl hydrocarbon receptor, knockout gene, CRISPR/Cas9, puromycin selection

Abstract

Mesenchymal stem cells (MSCs) have garnered significant attention in biomedical studies due to their remarkable properties, such as self-renewal, differentiation into diverse cell types and immune responses. The proliferation and differentiation of MSCs are significantly influenced by the ligand-dependent transcription factor known as the aryl hydrocarbon receptor (AhR). In order to understand the roles of Ahr in adipose-derived MSCs (AD-MSCs), we disrupted the Ahr gene in this work using CRISPR/Cas9 gene editing technology. The gRNA/Cas9 dual vector and donor vector were introduced into the AD-MSC cell line (PT-5006). Green fluorescent protein (GFP) expression and puromycin resistance were used to identify the transfected cells. AhR-KO cells were cloned and confirmed by PCR and sequencing. By using RT-qPCR, the expression levels of AhR and the Ahr-related gene Cyp1B1 were investigated. The results showed that the knocked out of AhR using CRISPR/Cas9, resulting significantly decreased expression of 7.69-fold for AhR and 3.70-fold for Cyp1B1 in the cells. These cell clones and CRISPR/Cas9 vectors could be used as tools to investigate the functions of AhR in both AD-MSCs and other cell types.

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Published

30-03-2024

How to Cite

Duc, L. H., Tien, V. T., & Nam, N. T. (2024). Knock out aryl hydrocarbon receptor gene in adipose-derived mesenchymal stem cell line (PT-5006) by CRISPR/Cas9. Vietnam Journal of Biotechnology, 22(1), 69–77. https://doi.org/10.15625/vjbt-20645

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