<p>Cytomix (pH 7.6) composition [14].</p>

Genetic manipulation of Toxoplasma gondii presents unique challenges due to its obligatory intracellular nature and relatively rapid growth. Electroporation is the main technique used to introduce genetic modifications into T. gondii. However, the existing protocols require an electroporation buffer comprised of eight components and involving multiple steps of preparation. Optimizing electroporation protocols, including a readily available buffer is crucial for achieving efficient transfection while simplifying the overall process. In this study, we present a modified Opti-MEM I based electroporation buffer that matches cytomix in performance with significantly reduced variability. We also develop a novel scoring method (etScore) to reproducibly quantify electroporation performance, combining transgene gene expression with cell viability. We also couple the experimental work with a corresponding systematic risk assessment and argue for routine use of such tools in similar contexts. We anticipate this protocol will make genetic modification of T. gondii more accessible to the international community, accelerating drug and vaccine research.刚地弓形虫（Toxoplasma gondii）的遗传操作因其专性胞内寄生特性与相对较快的增殖速度，面临独特的技术挑战。电穿孔（electroporation）是向刚地弓形虫引入遗传修饰的主流技术。然而现有方案所需的电穿孔缓冲液包含八种组分，且制备步骤繁杂。优化电穿孔方案——包括采用易于获取的缓冲液——对于实现高效转染同时简化整体流程至关重要。本研究中，我们开发了一种基于改良Opti-MEM I的电穿孔缓冲液，其性能可与cytomix相媲美，且批间差异显著降低。我们还开发了一种全新的评分方法（etScore），通过结合转基因表达与细胞活力，可重复地量化电穿孔效果。此外，我们将实验工作与对应的系统性风险评估相结合，并主张在类似研究场景中常规使用此类工具。我们预计该方案将使国际学界更易于开展刚地弓形虫的遗传修饰研究，从而加速药物与疫苗的研发进程。