Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes
Background: Models of muscle cell culture allow simple manipulation to optimize gene transfer experiments, offering a significant opportunity to model muscle effects by allowing precise control of the cell culture environment in a cost-effective manner. Often, gene transfer is performed in myoblast...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Mary Ann Liebert
2024-01-01
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| Series: | Re:GEN Open |
| Subjects: | |
| Online Access: | https://www.liebertpub.com/doi/full/10.1089/REGEN.2023.0026 |
| _version_ | 1797272653140066304 |
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| author | Amanda Sales Conniff Aneeshea Cason Juliana Gonzalez Alvarado Manya Bhandary Shreena Patel Julie Singh Richard Heller Loree C. Heller |
| author_facet | Amanda Sales Conniff Aneeshea Cason Juliana Gonzalez Alvarado Manya Bhandary Shreena Patel Julie Singh Richard Heller Loree C. Heller |
| author_sort | Amanda Sales Conniff |
| collection | DOAJ |
| description |
Background: Models of muscle cell culture allow simple manipulation to optimize gene transfer experiments, offering a significant opportunity to model muscle effects by allowing precise control of the cell culture environment in a cost-effective manner. Often, gene transfer is performed in myoblasts, which are then differentiated into myotubes, a skeletal muscle model. In this study, we aimed to compare the efficiency of direct transfection of differentiated myotubes by chemical and physical plasmid methods.
Methods: Differentiated myotubes were transfected with reporter plasmids. Transfection efficiency and cell death were quantified. Proinflammatory proteins are produced by cells and tissues after DNA transfection; therefore, the secretion of proinflammatory chemokines was quantified.
Results: In this study, we demonstrate that C2C12 myotubes can be effectively transfected using multiple methods. While a chemical method did not cause cell death, myotubes were not efficiently transfected. Electroporation delivery produced a higher transfection efficiency coupled with higher cell death. A subset of proinflammatory chemokine proteins was secreted, which replicated secretion by skeletal muscle.
Discussion: These results suggest that differentiated myotubes can be directly transfected invitro, which may represent a useful experimental model. However, more studies are needed to assess the value of this model in predicting invivo responses. |
| first_indexed | 2024-03-07T14:32:27Z |
| format | Article |
| id | doaj.art-e2e1260046754ded9ad3ab39116d0be1 |
| institution | Directory Open Access Journal |
| issn | 2766-2705 |
| language | English |
| last_indexed | 2024-03-07T14:32:27Z |
| publishDate | 2024-01-01 |
| publisher | Mary Ann Liebert |
| record_format | Article |
| series | Re:GEN Open |
| spelling | doaj.art-e2e1260046754ded9ad3ab39116d0be12024-03-06T04:00:40ZengMary Ann LiebertRe:GEN Open2766-27052024-01-01411810.1089/REGEN.2023.0026Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 MyotubesAmanda Sales Conniff0Aneeshea Cason1Juliana Gonzalez Alvarado2Manya Bhandary3Shreena Patel4Julie Singh5Richard Heller6Loree C. Heller7Department of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South FloridaDepartment of Medical Engineering, Morsani College of Medicine and College of Engineering, University of South Florida Background: Models of muscle cell culture allow simple manipulation to optimize gene transfer experiments, offering a significant opportunity to model muscle effects by allowing precise control of the cell culture environment in a cost-effective manner. Often, gene transfer is performed in myoblasts, which are then differentiated into myotubes, a skeletal muscle model. In this study, we aimed to compare the efficiency of direct transfection of differentiated myotubes by chemical and physical plasmid methods. Methods: Differentiated myotubes were transfected with reporter plasmids. Transfection efficiency and cell death were quantified. Proinflammatory proteins are produced by cells and tissues after DNA transfection; therefore, the secretion of proinflammatory chemokines was quantified. Results: In this study, we demonstrate that C2C12 myotubes can be effectively transfected using multiple methods. While a chemical method did not cause cell death, myotubes were not efficiently transfected. Electroporation delivery produced a higher transfection efficiency coupled with higher cell death. A subset of proinflammatory chemokine proteins was secreted, which replicated secretion by skeletal muscle. Discussion: These results suggest that differentiated myotubes can be directly transfected invitro, which may represent a useful experimental model. However, more studies are needed to assess the value of this model in predicting invivo responses.https://www.liebertpub.com/doi/full/10.1089/REGEN.2023.0026C2C12 myotubesplasmid DNAtransfectionelectroporationchemical methods |
| spellingShingle | Amanda Sales Conniff Aneeshea Cason Juliana Gonzalez Alvarado Manya Bhandary Shreena Patel Julie Singh Richard Heller Loree C. Heller Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes Re:GEN Open C2C12 myotubes plasmid DNA transfection electroporation chemical methods |
| title | Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes |
| title_full | Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes |
| title_fullStr | Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes |
| title_full_unstemmed | Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes |
| title_short | Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes |
| title_sort | direct plasmid dna transfection into differentiated mouse c2c12 myotubes |
| topic | C2C12 myotubes plasmid DNA transfection electroporation chemical methods |
| url | https://www.liebertpub.com/doi/full/10.1089/REGEN.2023.0026 |
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