Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts.

ABSTRACT: Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. T

SUBMITTER: Xie Z 

PROVIDER: S-EPMC5465212 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature