FIG 1.
Establishment of DGAT1-silenced cell lines using DGAT1-TALEN or lentivirus vector shRNA DGAT1. (A) Huh-7.5 cells were transfected with plasmids encoding a pair of DGAT1-targeting TALENs in combination with the reporter encoding H-2Kk. After the magnetic sorting of cells with TALEN-driven mutations, we analyzed 62 single-cell-derived colonies using the T7E1 assay. The region of the DGAT1 gene containing the TALEN target site was amplified by nested PCR and visualized by agarose gel electrophoresis. Representative data from the T7E1 assay are presented here. Circled numbers indicate clones that were selected for DGAT1 gene sequencing. (B) DNA sequences of the DGAT1-TALEN cell line. The sequence presented here is part of the DGAT1 gene of clone 13, one of the clones that are presented in panel A. Zinc finger nuclease recognition sites are underlined, and deleted bases are indicated by dashes. TGA, the premature stop codon, is shaded in gray. Clone 13 has a shifted open reading frame (ORF) or premature stop codon in the DGAT1 gene. (C) DGAT1 mRNA levels were determined by real-time quantitative PCR in DGAT1-silenced cell lines and standardized to β-actin mRNA levels (n = 3). N.D., not detected. (D) DGAT1 protein expression in DGAT1-silenced cell lines was assessed by immunoblotting. (E) Intracellular lipid droplet staining was performed using the BODIPY lipid probe 493/503 in the control and DGAT1-silenced cell lines. The scale bar represents 20 μm. The right panel represents flow cytometry analyses of the same cell lines.