Optimized electroporation buffer improves transfection and prime editing efficiency in adult bovine fibroblasts

For livestock breeding, using somatic cells from adult animals for gene editing and subsequent cloning allows the preservation and enhancement of superior traits from the parent directly in the offspring, while avoiding the loss of genetic gain that can occur through crossbreeding. However, primary cells generally more difficult to transfect and perform gene editing. To date, most related studies have used more vigorous fetal fibroblasts as donor cells, while using somatic cells from adult animals requires more post-editing screening efforts due to the low yield of edited cells. Here, we performed electroporation on adult bovine ear fibroblasts (BEFs) under various conditions such as electrical pulse settings, plasmid dosage, cell density, and concentration of electroporation buffer, and evaluated the transfect efficiency using flow cytometry analysis. We confirmed that the 270 V-10-10 program (270 V, 10 ms, 10 cycles) using 1.5 million cells and 5 µg of EGFP plasmid yielded the highest number of EGFP positive cells. Additionally, we used prime editor (PE) to edit the MSTN locus in BEFs. More importantly, we discovered that lowering the osmolarity of the electroporation buffer improves both electroporation and gene editing efficiency, which relate to the repression of cGAS-STING pathway. Our finding provides valuable references for using electroporation methods in adult bovine primary cell gene editing.