BMC Biotechnol. 2023 Aug 16;23(1):29.doi: 10.1186/s12896-023-00799-1.

Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system

Weiwei Liu  1 Xiaoguo Wang  1 Ruirong Liu  1 Yaya Liao  1 Zhiwei Peng  1 Haoyun Jiang  1 Qiqi Jing  1 Yuyun Xing  2



  • 1 State Key Laboratory of Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang, 330045, China.
  • 2 State Key Laboratory of Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang, 330045, China.

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Background: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips.

Methods: We firstly delivered a 13 kb Cas9-EGFP and a 3.5 kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13 kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector™ P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs.

Results: Notably lower transfection efficiencies were observed when delivering the 13 kb vector than delivering the 3.5 kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113 + CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13 kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113 + CA-137 and CA-137 + CA-137 programs showed significantly superior (P < 0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer.

Conclusions: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector™ system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector™ P3 buffer.

Keywords: 4D-Nucleofector™; Dual-electroporation program; Electroporation; Porcine fetal fibroblasts (PFFs).