Protocol for generating multiplexed prime-edited monoclonal cell lines in porcine fetal fibroblasts
Abstract
Porcine fetal fibroblasts (PFFs) serve as standard donor cells for generating cloned pigs, and prime editing (PE) enables precise genome modification. Here, we describe a protocol for generating multiplexed prime-edited monoclonal cell lines in PFFs. We describe steps for pegRNA/ngRNA design and screening, plasmid electroporation, nocodazole treatment, puromycin selection, and monoclonal isolation and genotyping. Although demonstrated by introducing three Alzheimer's disease-associated pathogenic mutations, the pipeline can be readily adapted to multiplex PE of other endogenous loci in porcine cells. For complete details on the use and execution of this protocol, please refer to Liu et al. 1
Fields of Study & Subjects
Pluripotent Stem Cells Research
Pancreatic function and diabetes
Tissue Engineering and Regenerative Medicine
Publication details
- Journal / source
- STAR Protocols
- Publisher
- Elsevier BV
- Volume
- 7
- Issue
- 3
- Pages
- 104691–104691
- Publication date
- 7 July 2026
- Language
- EN