Figure 3
From: Overexpression of hepatocyte EphA2 enhances liver-stage infection by Plasmodium vivax
Generation of the transgenic EphA2Extra-HC04 cell line using a CRISPR-Cas9 knock-in approach. (A) Introduction of HA-Extra to the HC-04 genome by Cas9-mediated homology directed repair (HDR). Genome integration was induced by Cas9 with sgRNA targeting the AAVS1 sequence. Homologous recombination was achieved using a linear donor containing the HA-Extra cassette flanked by AAVS1 homology arms. The figure was created with BioRender.com. (B) Left: PCR demonstrating the presence of the HA-Extra sequence in two clones of the transgenic EphA2Extra-HC04 cell line. The forward primer binds to a sequence upstream of the left homology arm; the reverse primer binds to the ligand-binding domain (LBD) of the insert, resulting in a 2143-bp amplicon. Primer binding sites are depicted in (A). Right: Western blot analysis of the EphA2Extra-HC04 cell line. Total cell lysates were analyzed with the indicated antibodies. β-actin was used as the loading control. (C) Representative IFA images of EphA2Extra-HC04 clones expressing HA-Extra on the cell surface. HA-Extra was visualized with a HA-tag antibody. Nuclei were stained with DAPI. Scale bar: 10 μm.
