دورية أكاديمية
أعرض في EDS

Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines.

التفاصيل البيبلوغرافية
العنوان: Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines.
المؤلفون: Hamada T; Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan., Yokoyama S; Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan., Akahane T; Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan., Matsuo K; Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan., Tanimoto A; Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
المصدر: International journal of molecular sciences [Int J Mol Sci] 2022 Dec 28; Vol. 24 (1). Date of Electronic Publication: 2022 Dec 28.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Basel, Switzerland : MDPI, [2000-
مواضيع طبية MeSH: Gene Editing*/methods , Glioblastoma*/genetics, Humans ; CRISPR-Cas Systems/genetics ; Ribonucleoproteins/genetics ; Ribonucleoproteins/metabolism ; Cell Line ; Nucleotides/metabolism
مستخلص: Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha ( PDGFRA ) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.
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فهرسة مساهمة: Keywords: Cas9 ribonucleoprotein; PDGFRA; cultured glioblastoma cell lines; single-nucleotide substitution; transfection methods
المشرفين على المادة: 0 (Ribonucleoproteins)
0 (Nucleotides)
تواريخ الأحداث: Date Created: 20230108 Date Completed: 20230110 Latest Revision: 20230111
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC9820287
DOI: 10.3390/ijms24010500
PMID: 36613947
قاعدة البيانات: MEDLINE
الوصف
تدمد:1422-0067
DOI:10.3390/ijms24010500