FIG 4.

Establishement of ApoE knockout human hepatic cell lines. (A) Schematic showing the CRISPR-Cas9 system and the target site for gRNA 299 in the ApoE gene locus used in this study. Protospacer adjacent motif (PAM) sequences are in orange. Fragments amplified by p1 with p2 and the AfeI restriction site (in aqua blue) were used in a restriction fragment length polymorphism gel assay. (B) Restriction fragment length polymorphism gel analysis. The CRISPR-Cas9 system was transiently expressed in Huh7.5 cells. After two rounds of subcloning and testing for protein expression, two monoclonal ApoE knockout cell lines were isolated and termed HuhdE1.3 and HuhdE1.5. The short lines indicate fragments generated by AfeI digestion. (C) Karyotype analysis of the Huh7.5 and ApoE knockout cell lines, Huh7dE1.3 and Huh7dE1.5. (D) Sequences of indel mutations identified in the ApoE gene using next-generation sequencing for each cell line. Red dashes, deletions; red bases, insertions.