Differential Gene Expression Induced by Different TLR Agonists in A549 Lung Epithelial Cells Is Modulated by CRISPR Activation of TLR10
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
A549 Challenge
2.2. sgRNAs and dCas9 Vectors
2.2.1. sgRNA Design
2.2.2. Validation of the sgRNAs
2.2.3. Plasmids and Cloning
2.3. Overexpression of the Endogenous TLR10 and Challenge of the Cells with Putative TLR10 Ligands
2.4. RNA Extraction and Reverse Transcription into Single-Stranded cDNA
2.5. Gene Expression Analyses
2.5.1. Quantitative PCR (qPCR) Array Screening
2.5.2. qPCR Expression Profiling
2.6. Protein Quantification
2.7. Western Blotting
2.8. Statistical Analysis
3. Results
3.1. CRISPR Activation of the TLR10
3.2. Differential Expression of Immune Associated Genes in the Challenged A549 Cells with Native Gene Expression
3.3. Differential Expression of Immune Associated Genes in the Challenged A549 Cells Overexpressing TLR10
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Knez, Š.; Narat, M.; Ogorevc, J. Differential Gene Expression Induced by Different TLR Agonists in A549 Lung Epithelial Cells Is Modulated by CRISPR Activation of TLR10. Biomolecules 2023, 13, 19. https://doi.org/10.3390/biom13010019
Knez Š, Narat M, Ogorevc J. Differential Gene Expression Induced by Different TLR Agonists in A549 Lung Epithelial Cells Is Modulated by CRISPR Activation of TLR10. Biomolecules. 2023; 13(1):19. https://doi.org/10.3390/biom13010019
Chicago/Turabian StyleKnez, Špela, Mojca Narat, and Jernej Ogorevc. 2023. "Differential Gene Expression Induced by Different TLR Agonists in A549 Lung Epithelial Cells Is Modulated by CRISPR Activation of TLR10" Biomolecules 13, no. 1: 19. https://doi.org/10.3390/biom13010019
APA StyleKnez, Š., Narat, M., & Ogorevc, J. (2023). Differential Gene Expression Induced by Different TLR Agonists in A549 Lung Epithelial Cells Is Modulated by CRISPR Activation of TLR10. Biomolecules, 13(1), 19. https://doi.org/10.3390/biom13010019