Optimized cuvette-based electroporation of buffalo and caprine zygotes supports the delivery of up to 21 kilobase plasmids

Abstract

The advent of precise genome editing technologies has revolutionized the capacity to manipulate animal genotypes and phenotypes, offering a targeted complement to conventional selective breeding approaches. Efficient delivery of exogenous DNA into livestock embryos remains a major technical bottleneck, particularly for large constructs, due to limitations of pronuclear microinjection and somatic cell nuclear transfer. In this study, we optimized a simplified cuvette-based electroporation method for the delivery of minicircles and shuttle plasmids (pGOF18) of different sizes (1.8–21.0 kbp) into bubaline and caprine zygotes at 10 h post-insemination. Embryos were evaluated for preimplantation developmental competence, total blastomere number, and transcriptional profiles of apoptotic (BAX, Caspase-9) and lineage-specific (OCT4, CDX2) genes. Electroporated embryos developed to the blastocyst stage with reporter-associated fluorescence, without significant differences in cleavage or blastocyst formation rates compared with controls. No significant differences in apoptotic genes expression were observed, indicating minimal cellular stress. Furthermore, pGOF18-electroporated embryos exhibited significant ( p  < 0.05) upregulation of OCT4 and CDX2 transcripts. These results demonstrate that optimized cuvette-based electroporation provides an efficient, reproducible, and embryo-tolerant strategy for introducing both large and small DNA constructs into goat and buffalo zygotes, supporting the generation of transgenic embryos while preserving preimplantation developmental competence.