Journal: PloS one

Article Title: ZO-1 knockout by TALEN-mediated gene targeting in MDCK cells: involvement of ZO-1 in the regulation of cytoskeleton and cell shape.

doi: 10.1371/journal.pone.0104994

Figure Lengend Snippet: Figure 1. Construction of TALENs and ZO-1 gene knockout in MDCK I and II cells. (A) TALEN binding sites in the ZO-1 gene. The left and right arms of TALEN targeting sites are indicated in blue and the spacer region is indicated in red. The initiation codon within the spacer region is highlighted. (B) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK II cells transfected with TALEN constructs for ZO-1 gene knockout. After transfection, cells were subcultured on filter inserts for 4 d before analysis. At the boundary of control and ZO-1 knockout cells, characteristic convex curves of cell–cell junctions are observed (arrows). (C) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK I cells transfected with TALEN constructs for ZO-1 gene knockout. Similar morphological changes of cell–cell junctions at the boundary of control and ZO-1 knockout cells were observed in MDCK I cells. Staining of ZO-3 was reduced in ZO-1 knockout cells (arrowheads). Scale bars, 10 mm. doi:10.1371/journal.pone.0104994.g001

Article Snippet: Construction of TALENs and establishment of knockout clones TALENs were constructed following the detailed instruction provided by the TALE Toolbox kit from the Zhang laboratory [27] (Addgene, #1000000019).

Techniques: TALENs, Gene Knockout, Binding Assay, Immunofluorescence, Transfection, Construct, Control, Knock-Out, Staining

Journal: PloS one

Article Title: ZO-1 knockout by TALEN-mediated gene targeting in MDCK cells: involvement of ZO-1 in the regulation of cytoskeleton and cell shape.

doi: 10.1371/journal.pone.0104994

Figure Lengend Snippet: Figure 4. Establishment of ZO-1 knockout clones in MDCK II cells. (A) Immunofluorescence microscopic analysis of ZO-1 in control (CTL) MDCK II cells and ZO-1 knockout clones (KO 1–3). ZO-1 staining was completely lost in ZO-1 knockout clones. Scale bar, 10 mm. (B) Immunoblots of ZO-1 and E-cadherin (E-cad) in control MDCK II cells and ZO-1 knockout clones. Knockout clones showed no detectable bands of ZO-1. (C) DNA sequences of TALEN targeting sites in each allele of ZO-1 knockout clones. One type of mutation was present in the alleles of ZO-1 knockout clone 1 (KO 1) and two types of mutations in the alleles of clones 2 and 3 (KO 2 and 3). Dashes indicate loss of nucleotides and green letters indicate additional nucleotides. Loss of initiating codon or frameshift were confirmed in all alleles. (D) Genomic PCR analysis of control and ZO-1 knockout clones using primers for TALENs and ZO-1 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs were detected in ZO-1 knockout clones. doi:10.1371/journal.pone.0104994.g004

Article Snippet: Construction of TALENs and establishment of knockout clones TALENs were constructed following the detailed instruction provided by the TALE Toolbox kit from the Zhang laboratory [27] (Addgene, #1000000019).

Techniques: Knock-Out, Clone Assay, Immunofluorescence, Control, Staining, Western Blot, Mutagenesis, TALENs, Stable Transfection, Expressing, Positive Control