Optimizing Electroporation Parameters for Efficient Delivery of Large Molecules into Pig Zygotes Using Fluorescent Dextrans from 3 to 2000 kDA

Electroporation has revolutionized gene transfer and gene editing, enabling efficient delivery of molecules into embryos, with significant implications for developmental biology and biomedical research. This study aimed to optimize electroporation parameters for enhancing the delivery of large molecules into pig zygotes. We investigated the effects of fluorescence-coupled dextran reporters (FDs) of sizes ranging from 3 to 2000 kiloDalton (kDA) along with the impact of poring and transfer polarity settings during electroporation, on molecule permeability. Additionally, we assessed the influence of voltage and the number of poring pulses on the delivery of 2000 kDa FDs and examined the permeability of pre-IVF embryos and zona pellucida-weakened post-IVF embryos to this FD. Our findings highlighted size-dependent effects on FD uptake, with reversing poring polarity increasing the influx of small molecules (3 kDa FDs). The delivery of 2000 kDa FDs was not influenced by increased poring number but it was significantly influenced by voltage, reaching its optimum at 40 V. Electroporation in pre-IVF embryos did not show significant variation across different voltages. However, voltages higher than 20 V negatively affected blastocyst development rates. Zona-weakening did not improve permeability for the 2000 kDa FD. This study offers valuable insights into refining electroporation techniques for delivering large molecules into pig zygotes and highlights the relevance of commercial fluorescence-coupled dextrans as useful tools for exploring permeability dynamics in electroporated zygotes.