使用 Lonza 4D-Nucleofector 系统高效递增大尺寸 Cas9-EGFP 载体至猪胎儿成纤维细胞。

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

机构信息

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

出版信息

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

DOI:10.1186/s12896-023-00799-1

PMID:37587435

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10428654/

Abstract

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.

摘要

背景

猪胎儿成纤维细胞（PFFs）是生成基因修饰猪的重要供体细胞，但 PFFs 的转染效率往往不尽人意，尤其是在递送大尺寸载体时。在这项研究中，我们旨在使用 Lonza 4D-Nucleofector™ 仪器和试剂盒优化递送大尺寸载体到 PFFs 中的转染条件。

方法

我们首先通过 Lonza 基本方案中推荐的 7 个程序，将一个 13kb 的 Cas9-EGFP 和一个 3.5kb 的 pMAX-GFP 载体递送到 PFFs 中。然后，我们测试了 6 个定制的双电穿孔程序，用于将 13kb 的质粒递送到 PFFs 中。此外，我们筛选了 Nucleofector™ P3 溶液的潜在替代电穿孔缓冲液。最后，我们使用不同的单和双电穿孔程序将三个靶向 Rosa26、H11 和 Cep112 基因座的 CRISPR/Cas9-sgRNA 递送到 PFFs 中。

结果

与使用单电穿孔程序将 3.5kb 载体递送到 PFFs 相比，明显观察到递送 13kb 载体时的转染效率较低。定制的双电穿孔程序 FF-113+CA-137 表现出比使用仪器（98.1%）或试剂盒（89.1%）的任何单电穿孔程序更高的转染效率，且对于 13kb 载体具有可接受的存活率。Entranster-E 缓冲液产生的转染效率和 24 小时存活率与 P3 溶液相当，因此可作为替代电穿孔缓冲液。在基因组编辑实验中，FF-113+CA-137 和 CA-137+CA-137 程序在仪器和试剂盒中均表现出明显优于（P<0.01）单电穿孔程序的效率。Entranster-E 缓冲液产生的插入缺失效率高于 P3 缓冲液。