Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts

Abstract

Background: Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. Results: Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 μg supercoiled plasmid DNA and 0.3 × 10 6 cells in 100 μl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. Conclusions: The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.

Application of transposon systems in the transgenesis of bovine somatic and germ cells

“…Following verification in somatic cells, embryo microinjection was performed to similarly verify that these systems worked reliably in germline cells. Firstly, in the bovine somatic cells, it was difficult to guarantee high efficiency and survival using primary cultured somatic cells instead of immortalized cell lines [ 18 ]. Therefore, methods that introduce viruses such as the adeno-associated virus (AAV), retrovirus, and lentivirus were implemented.…”

Application of Transposon Systems in The Transgenesis of Bovine Somatic And Germ Cells

“…After veri cation in the somatic cell, embryo microinjection was performed to verify that these systems worked reliably in germline cells. First, in the bovine somatic cell transfection experiment, it was di cult to guarantee high e ciency and survival using primary cultured somatic cells, and not immortalized cell lines (17). For this reason, methods of introducing viruses such as adeno-associated virus (AAV), retrovirus, and lentivirus were implemented.…”

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells

“…Cells were centrifugation at 1000 rpm for 3 min, and the cell pellet was resuspended into 250 µl of the electroporation medium. We compared electroporation efficiency of three buffers/media, including PBS, OptiMEM-GlutaMAX, and the Bio-Rad electroporation buffer with osmolality of 0.275, 0.290, and 0.430 (Osmol/kg) 26 , respectively. In an initial experiment, the highest transfection efficiency of iPS cells were reached by OptiMEM-GlutaMAX medium (Supplementary Figure S1).…”