Author:
Liu Weiwei,Wang Xiaoguo,Liu Ruirong,Liao Yaya,Peng Zhiwei,Jiang Haoyun,Jing Qiqi,Xing Yuyun
Abstract
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.
Funder
National Science and Technology Major Project
National Natural Science Foundation of China
Publisher
Springer Science and Business Media LLC
Reference30 articles.
1. Walters EM, Wells KD, Bryda EC, Schommer S, Prather RS. Swine models, genomic tools and services to enhance our understanding of human health and diseases. Lab Anim. 2017;46(4):167–72.
2. Flisikowska T, Kind A, Schnieke A. Pigs as models of human cancers. Theriogenology. 2016;86(1):433–7.
3. Galli C, Lazzari G, 25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER. Current applications of SCNT in advanced breeding and genome editing in livestock. Reproduction. 2021;162(1):F23–F32.
4. Zhang J, Khazalwa EM, Abkallo HM, Zhou Y, Nie X, Ruan J, Zhao C, Wang J, Xu J, Li X, et al. The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research. J Genet Genomics. 2021;48(5):347–60.
5. Liu T, Dou H, Xiang X, Li L, Li Y, Lin L, Pang X, Zhang Y, Chen Y, Luan J, et al. Factors determining the efficiency of porcine somatic Cell Nuclear transfer: Data Analysis with Over 200,000 reconstructed embryos. Cell Reprogram. 2015;17(6):463–71.