Figure 3

Establishment of the homozygous Nrf1α^−/− knockout monoclonal cell line by using TALENs.

(a) HepG2 cells were co-transfected with TALEN-left (1.5 μg) and TALEN-right (3 μg) constructs (along with 0.5 μg of a GFP-expressing plasmid to verify the transfection efficacy). The cells were selected by 2.5 μg/ml puromycin for 48 h before being subjected to the single cell cloning in 96-well plates, in order to establish the homozygous Nrf1α^−/− knockout monoclonal cell line. (b) The genomic DNA from the individual cell clones served as a template of PCR to amplify TALENs-recognized region of Nrf1. The sequencing result revealed overlapped peaks of distinct nucleotide waves starting around the site responsible for the initial translation of Nrf1α. (c) A nucleotide alignment of human wild-type (WT) Nrf1 and its frameshift mutants around TALENs-target sequences. The deletion nucleotides were represented by dashed dots. The putative cDNA codons were placed in the black backgrounds. (d) Different mRNA levels of Nrf1 in TALENs-mediated mutant cell lines (called HA to HI) and wild-type HepG2 cells were measured by quantitative real-time PCR. The results were calculated as a fold change (mean ± S.E.) of Nrf1 transcriptional expression. Significant decreases (*p < 0.05, **p < 0.01, n = 9) are indicated, relative to the wild-type control value of 1 measured from HepG2 cells. (e,f) Total lysates of each cell lines that had been treated with MG132 or untreated were subjected to protein separation by 8% Laemmli SDS-PAGE gels running in the pH 8.9 Tris-glycine buffer, followed by immunoblotting with Nrf1 antibodies to determine the protein expression patterns. The standard (Std) sample was made from human Nrf1-overexpressing cells. (g) The lysates (re-grouped into 3 blocks) were subjected to further protein resolution by 4–12% LDS-NuPAGE gels running in the pH 7.3 MES buffer. (h,i) The homozygous Nrf1α^−/−-specific knockout monoclonal cell lines (e.g. HEA157) were established on the base of the heterozygous mutant HA cells. The cell lines were further identified by sequencing of TALENs-target genomic DNA (c), quantitative real-time PCR (d) and western blotting with different two antibodies against Nrf1 that had been isolated by 10% SDS-PAGE gels (h,i).