Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter | ||
| Trends in Pharmaceutical Sciences and Technologies | ||
| مقاله 3، دوره 9، شماره 4، زمستان 2023، صفحه 253-260 اصل مقاله (525.46 K) | ||
| نوع مقاله: Original Article | ||
| شناسه دیجیتال (DOI): 10.30476/tips.2023.99754.1208 | ||
| نویسندگان | ||
| Arezoo Jokar1؛ Shayan Khalili Alashti1، 2؛ Maryam Baneshi1؛ Ali Rastegarian3؛ Kimia Sadat Hashemi4؛ Mohadese Koohi Aliabadi5؛ Mojtaba Kashfi6؛ amir savardashtaki* 7 | ||
| 1Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran | ||
| 2Epilepsy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran | ||
| 3Assistant Professor of Periodontics, Shiraz University of Medical Sciences, Shiraz, Iran | ||
| 4Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran | ||
| 5Faculty of Interdisciplinary Science and Technology, Tarbiat Modares University, Tehran, Iran | ||
| 6Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran | ||
| 7Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran | ||
| چکیده | ||
| Background and aim: CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. Method: This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 cells were cultured, synchronized, and transfected with sgRNAs using lipofectamine. Successful transfections were confirmed by fluorescence microscopy. Clonal expansion followed, with DNA extracted and genotyped using PCR and Sanger sequencing. Bisulfite conversion analyzed DNA methylation, employing restriction enzymes for CpG site analysis. Statistical significance (p≤0.05) was assessed using SPSS software. Results: The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. Conclusion: The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted. | ||
| کلیدواژهها | ||
| KHDC3L gene؛ PEG3 promoter؛ CRISPR/Cas9؛ gene knock-out | ||
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