Comparison of different electroporation parameters on transfection efciency of sheep testicular cells

Niakan, Sarah. and Heidari, Banafsheh. and Akbari, Ghasem. and Nikousefat, Zahra. (2016) Comparison of different electroporation parameters on transfection efciency of sheep testicular cells. Cell Journal, 18 (3). pp. 425-437.

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Abstract

Objective: Electroporation can be a highly efficient method for introducing the foreign genetic materials into the targeted cells for transient and/or permanent genetic modification. Considering the application of this technique as a very efficient method for drug, oligonucleotide, antibody and plasmid delivery for clinical applications and production of transgenic animals, the present study aimed to optimize the transfection efficiency of sheep testicular cells including spermatogonial stem cells (SSCs) via electroporation. Materials and Methods: This study is an experimental research conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran) from September 2013 to March 2014. Following isolation and propagation of one-month lamb testicular cells (SSCs and somatic testicular cells including; Sertoli, Leydig, and myoid cells), the effect of different electroporation parameters including total voltages (280, 320, and 350 V), burst durations (10, 8, and 5 milliseconds), burst modes (single or double) and addition of dimethyl sulfoxide (DMSO) were evaluated on transfection efficiency, viability rate and mean fluorescent intensity (MFI) of sheep testicular cells. Results: The most transfection effciency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a signifcantly inverse correlation between transfection effciency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and lowest viability rates of testicular cells were demonstrated in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Addition of DMSO to transduction medium in all groups signifcantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had the most?uorescence intensity in 320 V/double burst/DMSO positive. However, myoid and Leydig cells showed the maximum expression in 320 V/single burst and/or 350 V/double burst/DMSO positive. Conclusion: We optimized the electroporation method for transfection of sheep testicular cells and recommended the application of 320 V/8 milliseconds/single pulse/DMSO negative for transduction of plasmid vector into these cells. Among testicular cells, the most external gene expression was demonstrated in SSC population.

Item Type: Article
Additional Information: cited By 0
Uncontrolled Keywords: Electroporation, Sheep, Spermatogonial Stem Cells ,Testicular Cells, Transfection
Subjects: WJ Urogenital System
QU Biochemistry
Divisions: Reserach Vice-Chancellar Department > Cellular and Molecular Research Center
Depositing User: zahra bagheri .
Date Deposited: 15 Jul 2017 09:38
Last Modified: 30 Jan 2018 04:27
URI: http://eprints.skums.ac.ir/id/eprint/1016

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