Figure 1.
Scheme of the WT exon 4 showing the position of the different restriction enzyme sites and corresponding TALEN pairs.
The murine Ugt1 exon 4 and flanking introns is shown in the figure. The relative position of the NlaIV, NcoI, BstXI and MnlI restriction sites is indicated, as well as the left and right arm of each TALEN pair. Exonic and intronic sequences are indicated in yellow and white, respectively.
Figure 2.
Evaluation of TALEN activity using a Luciferase-reporter plasmid.
A) Scheme of the TALEN reporter plasmids and strategy. The Ugt1 exon 4 and flanking introns were introduced into the polylinker of the pGL3 (Linker) plasmid. This plasmid contains a 548 bp-repeated region of the luciferase cDNA. DSB induced by TALENs stimulates homologous recombination between the repeated luciferase regions and results in the recovery of luciferase enzyme activity. B) The reporter plasmid without TALENs showed no luciferase activity. 0.4 µg, 0.8 µg and 1.6 µg of pGL3-control and pGL3 (Linker) plasmids (lanes 1–3 and 4–6, respectively) were co-transfected with the 40 ng of renilla-luc plasmid into HEK293 cells. The pGL3 (Linker) plasmids showed no luciferase activity (lanes 4–6). C) Scheme of the reporter plasmid used in the TALEN assays. The figure shows the schemes of the pGL3-control, pGL3 (Linker) and pGL3-Ugt1(Ex4)WT plasmids. D) The NcoI TALEN pair shows the highest activity. The MnlI, NcoI, NlaIV and BstXI TALEN pairs were co-transfected into HEK293 cells together with the reporter pGL3-Ugt1 (Ex4)WT and renilla luc plasmids. Increasing amounts of the TALEN plasmids were co-transfected (200 ng for lanes 2, 6, 10 and 14; 400 ng for lanes 3, 7, 11, and 15; 800 ng for lanes 4, 8, 12, and 16). Activity was normalized to lanes 1, 5, 9, and 13, which contain pGL3 (linker) co-transfected with the higher amount of the respective TALEN pair.
Figure 3.
Transient transfection of NcoI TALENs into N-Muli murine hepatoma cells.
A) Determination of TALEN activity by RE-digestion of genomic PCR product. N-Muli cells were transfected with the NcoI TALEN pair together with a GFP plasmid. GFP positive cells were sorted, genomic DNA prepared and PCR amplified. PCR products were digested or not with NcoI, and the digestion product run in a 2% agarose gel. The arrow indicates the NcoI-resistant fraction of the PCR product to the NcoI-TALEN transfected cells (Lane 5). B) Confirmation of the presence of NcoI-resistant fragment. The agarose regions corresponding to the full-length undigested fragment of the gel shown in Panel A (GFP and NcoI-TALEN, lanes 4 and 5, respectively) were cut and DNA purified. A nested PCR reaction using internal primers was performed, and the PCR product was digested or not with NcoI (lanes 3, 6 and 2, 5, respectively). The percentage of uncut fragment is indicated bellow the gel. C) INDEL analysis of NcoI-TALEN N-Muli treated cells. The NcoI-resistant fragment of Panel A, Lane 5 was purified, cloned into a pUC19 vector and the obtained clones were sequenced. The left and right arms of the TALEN pair are indicated in green and red, respectively. The NcoI restriction enzyme site is indicated in blue. The deletions are indicated as black dashes. The type and length of the modification are indicated on the right of each sequence (Δ indicates deletion, and + base substitution, indicated by small orange caps).
Figure 4.
Analysis of N-Muli Ugt1-mutant cell clones.
A) and B) Schemes of the PCR fragments showing the position of the PCR primers and restriction sites. The position of the MnlI and NcoI sites are indicated (Panels A and B, respectively), as well as the expected length of the digestion products. C) and D) Restriction enzyme assay to detect INDELs. DNA from antibiotic-resistant clones (Panels C and D, from MnlI and NcoI TALEN-transfected cells, respectively) was prepared, PCR amplified, digested or not with MnlI and NcoI and the products run in a 1% agarose gel. The expected and obtained gel bands are indicated with arrows. N-Muli corresponds to control DNA, Mnl4, Mnl20, Nco25 and Nco20 to antibiotic-resistant clones that were transfected with the respective TALEN pairs.
Table 1.
Generation of Ugt1 N-Muli mutant cells by TALENs.
Figure 5.
Molecular analysis of cell clones.
A) INDEL analysis of MnlI-TALEN and NcoI-TALEN N-Muli treated cells. The left and right arms of the TALEN pair are indicated in green and red, respectively. The restriction enzyme sites are indicated in blue. Deletions are indicated as dashes. The length of the deletions is indicated on the right of each sequence. B) Western blot analysis of the isolated cell clones. Proteins were prepared from the isolated cell clones and analyzed by Western blot using an anti-Ugt1 antibody. Lanes 1 and 2 correspond to protein extracts prepared from WT and mutant livers [13]. Lane 3 corresponds to untreated N-Muli cells. The arrow indicates the band corresponding to Ugt1. β-tubulin was used to normalize for protein loading. C) Ugt1 glucuronidation activity of the isolated cell clones. Ugt1 glucuronidation activity was determined by the UGT-Glo Assay as described in the Materials and Methods section. The activity determination was determined with microsomes prepared from wt and Ugt mutant mouse liver (1 µg), from wt N-Muli cells (10 µg) and from the isolated cell clones (10 µg, Mnl20, Nco20 and Nco25), as the percentage of luminescent substrate consumed. “+cntrl” indicates microsomes provided by the manufacturers (Promega) as positive control of the experiment. The experiment was repeated twice, and the mean of both experiments is shown.