Establishment of an electroporation-mediated gene delivery system in porcine spermatogonial stem cells
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Spermatogonial stem cells (SSCs) are a useful tool for the generation of genetically modified transgenic sperm. As a result, the transfer of specific genes into the cytoplasm of SSCs is crucial for the successful generation of transgenic sperm. Here, we report electroporation conditions optimized for SSCs derived from the porcine testis. The highest transfection efficiency and cell viability were observed in porcine SSCs transfected with 1 μg transgenic vector with a single electric pulse from an electroporator at a voltage of 100 V and a capacitor setting of 250 μF. The transfection efficiency and cell viability were constant regardless of the size of the transgenic vector. Furthermore, we did not detect loss of spermatozoa differentiation potential in the transfected porcine SSCs. From these results, we confirm that this electroporation-based gene delivery system can effectively introduce foreign DNA into the genome of porcine SSCs without any loss of the original porcine SSC characteristics, which will be important in the generation of mosaicism-free transgenic pigs produced from transgenic porcine sperm.
KeywordsPig Spermatogonial stem cells Transfection Gene delivery system Electroporation
We thank Joohyeong Lee and Keun Cheon Kim for assistance of IVF and cryostat sectioning, respectively.
This research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bioindustry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (IPET117042-3), Republic of Korea.
Compliance with ethical standards
All of the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Kangwon National University (Approval No. KW-131106-1) and were performed in accordance with the Animal Care and Use Guidelines of Kangwon National University (Kangwon-do, South Korea).
Conflict of interest
The authors declare that they have no conflict of interests.
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