Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation
| Autor: | Hiroshi Kitamura, Takeshi Ishino, Rika Numano, Yoshinori Shimamoto, Hirofumi Kurita, Rikio Kirisawa |
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| Rok vydání: | 2020 |
| Předmět: |
Male
electroporation endocrine system animal structures Swine viruses Green Fluorescent Proteins Transfection medicine.disease_cause Biochemistry sperm Green fluorescent protein Flow cytometry law.invention Transduction (genetics) Plasmid swine cell law medicine Animals Escherichia coli Cells Cultured Full Paper General Veterinary medicine.diagnostic_test Chemistry Electroporation fungi Fibroblasts Spermatozoa Molecular biology Recombinant Proteins Mice Inbred C57BL embryonic structures Recombinant DNA Cattle bovine cell Plasmids |
| Zdroj: | The Journal of Veterinary Medical Science |
| ISSN: | 1347-7439 0916-7250 |
| Popis: | Instrument cost is a major problem for the transduction of DNA fragments and proteins into cells. Water-in-oil droplet electroporation (droplet-EP) was recently invented as a low-cost and effective method for the transfection of plasmids into cultured human cells. We here applied droplet-EP to livestock animal cells. Although it is difficult to transfect plasmids into bovine fibroblasts using conventional lipofection methods, droplet-EP enabled us to introduce an enhanced green fluorescent protein (EGFP)-expressing plasmid into bovine earlobe fibroblasts. The optimal transfection condition was 3.0 kV, which allowed 19.1% of the cells to be transfected. For swine earlobe fibroblasts, the maximum transfection efficacy was 14.0% at 4.0 kV. After transfection with droplet-EP, 69.1% of bovine and 76.5% of swine cells were viable. Furthermore, droplet-EP successfully transduced Escherichia coli recombinant EGFP into frozen-thawed bovine sperm at 1.5 kV. Flow cytometry analysis revealed that 71.5% of spermatozoa exhibited green fluorescence after transfection. Overall, droplet-EP is suitable for the transfection of plasmids and proteins into cultured livestock animal cells. |
| Databáze: | OpenAIRE |
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