Comparison of Different Electroporation Parameters on Transfection Efficiency of Sheep Testicular Cells
Journal Title: Cell Journal(Yakhteh) - Year 2016, Vol 18, Issue 3
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 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 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 significantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had 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 negative for transduction of plasmid vector into these cells. Among testicular cells, the most external gene expression was demonstrated in SSC population.
Authors and Affiliations
Banafsheh Heidari
Keywords
Related Articles
- EP ID EP553212
- DOI 10.22074/cellj.2016.4571
- Views 162
- Downloads 0
Synthesis and Characterization of PMBN as A Biocompatible Nanopolymer for Bio-Applications
Objective: Poly [2-methacryloyloxyethyl phosphoryl choline (MPC)-co-n-buthyl methacrylate (BMA)-co-p-nitrophenyl-oxycrabonyl poly ethylene glycol-methacrylate (MEONP)] (PMBN), a biocompatible terpolymer, is a unique poly...
Boron Induces Early Matrix Mineralization via Calcium Deposition and Elevation of Alkaline Phosphatase Activity in Differentiated Rat Bone Marrow Mesenchymal Stem Cells
Objective Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular...
A Bibliometric Analysis Of Publications On Pluripotent Stem Cell Research
Objective Human pluripotent stem cells are self-renewing cells with the ability to differentiate into a variety of cells and are viewed to have great potential in the field of regenerative medicine. Research in pluripote...
Stress Hormone and Oxidative Stress Biomarkers Link Obesity and Diabetes with Reduced Fertility Potential
Objective: Tilting the balance in favor of antioxidant agents could increase infertility problems in obese and diabetic individuals. The aim of this study was to evaluate oxidative stress status in semen of men with type...
FACT or PACT: A Comparison between Free-Acrylamide and Acrylamide-Based Passive Sodium Dodecyl Sulfate Tissue Clearing for whole Tissue Imaging
Major biological processes rely on the spatial organization of cells in complex, highly orchestrated three-dimensional (3D) tissues. Until the recent decade, most of information on spatial neural representation primarily...