دورية أكاديمية
أعرض في EDS

Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.

التفاصيل البيبلوغرافية
العنوان: Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.
المؤلفون: Pavlin M; Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Vrazov Trg 2, 1000, Ljubljana, Slovenia. mojca.pavlin@mf.uni-lj.si.; Group for Nano and Biotechnological Applications, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia. mojca.pavlin@mf.uni-lj.si., Škorja Milić N; Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia.; Institute of Anatomy, Faculty of Medicine, University of Ljubljana, Korytkova 2, Ljubljana, Slovenia., Kandušer M; Group for Nano and Biotechnological Applications, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia.; Pharmacy Institute, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia., Pirkmajer S; Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia. sergej.pirkmajer@mf.uni-lj.si.
المصدر: Biomedical engineering online [Biomed Eng Online] 2024 May 16; Vol. 23 (1), pp. 47. Date of Electronic Publication: 2024 May 16.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: BioMed Central Country of Publication: England NLM ID: 101147518 Publication Model: Electronic Cited Medium: Internet ISSN: 1475-925X (Electronic) Linking ISSN: 1475925X NLM ISO Abbreviation: Biomed Eng Online Subsets: MEDLINE
أسماء مطبوعة: Original Publication: London : BioMed Central, [2002-
مواضيع طبية MeSH: Electroporation*/methods , RNA, Small Interfering*/genetics , Muscle Fibers, Skeletal*/metabolism , Muscle Fibers, Skeletal*/cytology , Gene Silencing* , Cell Differentiation*, Humans ; Cell Survival ; Electrophoresis ; Transfection/methods
مستخلص: Background: Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines.
Results: We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved.
Conclusions: The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.
(© 2024. The Author(s).)
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معلومات مُعتمدة: P1-0055, J3-3077, MRIC UL IP-0510 Javna Agencija za Raziskovalno Dejavnost RS; P3-0043, J7-3153, J7-8276 Javna Agencija za Raziskovalno Dejavnost RS; P3-0043, J7-3153, J7-8276 Javna Agencija za Raziskovalno Dejavnost RS
فهرسة مساهمة: Keywords: Electrophoresis; Electroporation; Electrotransfection; Gene silencing; Mechanisms; Primary human myotubes; siRNA
تواريخ الأحداث: Date Created: 20240515 Date Completed: 20240516 Latest Revision: 20240518
رمز التحديث: 20240518
مُعرف محوري في PubMed: PMC11097476
DOI: 10.1186/s12938-024-01239-7
PMID: 38750477
قاعدة البيانات: MEDLINE
الوصف
تدمد:1475-925X
DOI:10.1186/s12938-024-01239-7