Abstract
Electroporation is an effective technique of transfection, but its efficiency depends on the optimization of various parameters. In this study, a simplified and efficient method of gene manipulation was standardized through electroporation to introduce a recombinant green fluorescent protein (GFP) construct as well as RNA-inhibitors in intact mouse follicles, oocytes and early embryos, where various electroporation parameters like voltage, pulse number and pulse duration were standardized. Electroporated preantral follicles were cultured further in vitro to obtain mature oocytes and their viability was confirmed through the localization of a known oocyte maturation marker, ovastacin, which appeared to be similar to the in vivo-derived mature oocytes and thus proved the viability of the in vitro matured oocytes after electroporation. Standardized electroporation parameters, i.e., three pulses of 30 V for 1 millisecond at an interval of 10 s, were applied to manipulate the expression of mmu-miR-26a in preantral follicles through the electroporation of miR inhibitors and mimics. The TUNEL apoptosis assay confirmed the normal development of the electroporated embryos when compared to the normal embryos. Conclusively, for the first time, this study demonstrated the delivery of exogenous oligonucleotides into intact mouse follicles, oocytes and embryos without hampering their zona pellucida (ZP) and further development.
Highlights
Electroporation is an effective non-viral technique that breaches the cell membrane [1]and continues to be accepted as a delivery system at the cellular level for many molecules like DNA, dsRNA, siRNA, mRNA, proteins, peptides, antibodies and drugs [2,3,4,5,6,7,8,9,10,11]
In order to measure electroporation transfection efficiency in follicles, oocytes and different embryonic stages, the strategy shown in Figure 1c was employed
Following transfection with pEGFP-C2 plasmid, the intensity of EGFP expression was measured under fluorescence microscopy in follicles, oocytes and different embryonic stages by merging all Z-stack slices after 48 h of electroporation
Summary
Electroporation is an effective non-viral technique that breaches the cell membrane [1]and continues to be accepted as a delivery system at the cellular level for many molecules like DNA, dsRNA, siRNA, mRNA, proteins, peptides, antibodies and drugs [2,3,4,5,6,7,8,9,10,11]. Various techniques have been attempted to improve the transfection efficiencies in various cell types by optimizing the transfection parameters, including voltage, pulse duration, number of pulses and pulse interval. The fluorescence expression property of green fluorescent protein (GFP) was explored for evaluation and assessment of transfection efficiency. GFP acts as an energy-transfer acceptor and transduces the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. We optimized electroporation parameters and demonstrated delivery of DNA/RNA across the zona pellucida (ZP), along with increased molecular uptake, while maintaining cell viability. Ovarian follicle development commences with the formation of primordial follicles, which further advances to subsequent stages by changing the shape and expanding the granulosa cell network around the oocyte
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