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

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

SUBMITTER: Liu W 

PROVIDER: S-EPMC10428654 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature