Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts
Abstract Background Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited p...
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| Format: | Article |
| Language: | English |
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BMC
2020-02-01
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| Series: | BMC Molecular and Cell Biology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12860-020-00254-5 |
| _version_ | 1818131619987324928 |
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| author | Iqbal Hyder Shahin Eghbalsaied Wilfried A. Kues |
| author_facet | Iqbal Hyder Shahin Eghbalsaied Wilfried A. Kues |
| author_sort | Iqbal Hyder |
| collection | DOAJ |
| description | Abstract Background Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. Results Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 μg supercoiled plasmid DNA and 0.3 × 106 cells in 100 μl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. Conclusions The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species. |
| first_indexed | 2024-12-11T08:23:49Z |
| format | Article |
| id | doaj.art-0db1923dd82d451394e8ff683178496a |
| institution | Directory Open Access Journal |
| issn | 2661-8850 |
| language | English |
| last_indexed | 2024-12-11T08:23:49Z |
| publishDate | 2020-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Molecular and Cell Biology |
| spelling | doaj.art-0db1923dd82d451394e8ff683178496a2022-12-22T01:14:36ZengBMCBMC Molecular and Cell Biology2661-88502020-02-012111810.1186/s12860-020-00254-5Systematic optimization of square-wave electroporation conditions for bovine primary fibroblastsIqbal Hyder0Shahin Eghbalsaied1Wilfried A. Kues2Institute of Farm Animal Genetics, Friedrich-Loeffler-InstituteInstitute of Farm Animal Genetics, Friedrich-Loeffler-InstituteInstitute of Farm Animal Genetics, Friedrich-Loeffler-InstituteAbstract Background Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. Results Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 μg supercoiled plasmid DNA and 0.3 × 106 cells in 100 μl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. Conclusions The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.http://link.springer.com/article/10.1186/s12860-020-00254-5FibroblastsElectroporationTransfection efficiencySquare wave pulse |
| spellingShingle | Iqbal Hyder Shahin Eghbalsaied Wilfried A. Kues Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts BMC Molecular and Cell Biology Fibroblasts Electroporation Transfection efficiency Square wave pulse |
| title | Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts |
| title_full | Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts |
| title_fullStr | Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts |
| title_full_unstemmed | Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts |
| title_short | Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts |
| title_sort | systematic optimization of square wave electroporation conditions for bovine primary fibroblasts |
| topic | Fibroblasts Electroporation Transfection efficiency Square wave pulse |
| url | http://link.springer.com/article/10.1186/s12860-020-00254-5 |
| work_keys_str_mv | AT iqbalhyder systematicoptimizationofsquarewaveelectroporationconditionsforbovineprimaryfibroblasts AT shahineghbalsaied systematicoptimizationofsquarewaveelectroporationconditionsforbovineprimaryfibroblasts AT wilfriedakues systematicoptimizationofsquarewaveelectroporationconditionsforbovineprimaryfibroblasts |