(10) succeeded in transfecting of cock SSCs using a single pulse of 270 V/80 milliseconds and reported the 29.37% of positive cells with 69.86% viability rate. 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 efficiency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a significantly inverse correlation between transfection efficiency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and least expensive viability rates of testicular cells were exhibited in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Ad- dition of DMSO to transduction medium in all groups significantly D-γ-Glutamyl-D-glutamic acid decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs experienced the most fluorescence 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 unfavorable for transduction of plasmid vector into these cells. Among testicular cells, the most external gene expression was exhibited in SSC populace. and for clinical applications (17,18). Many studies have now shown that plasmid electro-transfer can lead to a long-lasting therapeutic effect in some diseases, such as cancer, blood disease, or muscle mass ischemia (22,26). There are several reports of successful transfection of different cells including heart myoblast cells (27), mammary epithelial cells (28), retinal and iris pigment epithelial cells (29), dental pulp stem cells (30), adipose and mesenchymal stem cells (31), embryonic and adult neural stem cells (32), etc., through electroporation. Since stem cells are considered to be D-γ-Glutamyl-D-glutamic acid able to propagate infinitely, transduction and growth of transfected SSCs are necessary for development assay, fertility preservation, disease modeling, male infertility treatment, and production of transgenic animals (1,2). Due to the low transfection efficiency of electroporation in spite of its advantages and the importance of this germ collection, considerable efforts should be performed to establish more efficient protocols for transfected SSCs collection generation. The transfection efficiency of electroporation is usually highly dependent on the cell environment and conditions in which electric pulse are applied. In some cases, electroporation parameters utilized under one condition for transfecting a particular cell line may not necessarily be optimal for another cell collection. Thus, the transfection protocol should be specifically optimized for each condition and each type of cell collection. D-γ-Glutamyl-D-glutamic acid In present study, we investigated the effect of electroporation parameters including total volt, burst duration, quantity of bursts on total transfection efficiency, viability rate and imply fluorescence intensity (MFI) of testicular cells D-γ-Glutamyl-D-glutamic acid including SSCs. In order to improvement of the transfection efficiency and increasing the permeability of cell membrane, we used dimethyl sulfoxide (DMSO) as a transfection enhancing reagent to transduction medium and evaluated above parameters in all groups. Materials and Methods All experimental procedures were carried out with the recommendations in the guidelines for the care and use of animals by Avicenna Research Institute Animal Care and Use Committee. Cell isolation and preparation This study is an experimental research that conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran) from September 2013 to March 2014. Testis samples were collected from sheep between 1 to 3 months of age at a commercial slaughterhouse HGFR and transported to the lab in transition media [phosphate buffered saline (PBS) supplemented with 100 IU/ml penicillin (GibcoBRL, USA) and 100 g/m1 streptomycin (GibcoBRL, USA)] in an ambient heat. Testicular cell suspensions were prepared using a protocol previously explained (33). Briefly, after collection of testes and removing the tunica albuginea and visible connective tissues, the testes samples were minced with fine scissors and transferred into the Dulbeco Modified Essential Medium (DMEM, GibcoBRL, USA) supplemented with 14 mol/L NaHCO (Sigma, Germany), 10 l/ml nonessential amino acids (NEAA, Sigma, Germany), 50 IU/ml penicillin and 50 mg/ml streptomycin for 5-8 moments. The SSCs were isolated through two-step D-γ-Glutamyl-D-glutamic acid digestion method by collagenase type 1 (1 mg/ml, Gibco Burlington, Canada) and trypsin-EDTA (0.25%/1 mM, Sigma, Germany), respectively. The suspension was filtered successively through 60 m nylon mesh.
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