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. 2015 Jun 10;4(8):913–926. doi: 10.5966/sctm.2015-0030

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Figure 4. — TALEN-mediated single-base change of gfp gene on genomic DNA. (A): Strategy of single-base modification in gfp gene. The ssODN template with a single base change from the wild-type sequence introduces a stop codon and a BfaI restriction enzyme digestion site. The second ssODN removes the stop codon and adds a new SacI restriction enzyme digestion site. (B): Fluorescent-activated cell sorting (FACS) analysis of fluorescence cell population 3 days after either transfection of ssODN-1 and eukaryotic expression plasmids encoding the TALEN pair (transfection; BII) or transfection of ssODN-1 followed by injection of TALEN proteins by P. aeruginosa (transfection-injection; BIII). As a control, an untreated EB5 cell is shown (BI). Percentages of the GFP-negative cells in the whole population are shown. (C): A 350-bp fragment encompassing the TALEN-targeting region was amplified by polymerase chain reaction (PCR) from GFP-negative EB5 cells that were FACS-sorted after either transfection of ssODN-1 and TALEN coding plasmids or ssODN-1 transfection followed by TALEN protein injection. The PCR products were subjected to 2% agarose electrophoresis with (+) or without (−) digestion by BfaI restriction enzyme. An uninfected EB5 cell was used as the control. M represents the DNA marker. The percentage of mutation was calculated using ImageJ. (D): Single cell cloning of EB5 with desired single-base change in the gfp gene. The gfp fragments were PCR amplified from 12 cell lines obtained by single cell cloning and subjected to 2% agarose electrophoresis after digestion by the BfaI restriction enzyme. Two desired cell lines, numbers 4 and 6, were obtained. (E): FACS analysis of fluorescence cell population 3 days after transfection of ssODN-2 and injection of TALEN proteins by P. aeruginosa. As a control, gfp-silenced EB5 cells (EB5-Mut1) were injected with the TALEN proteins only. The percentage of the GFP-positive cells in the whole population are shown. (F): After transfection of ssODN-2 and TALEN protein injection into EB5-Mut1, GFP-positive cells were FACS-sorted, and a 350-bp fragment encompassing the TALEN-targeting region was amplified by PCR. The PCR products were digested with (+) or without (−) the SacI restriction enzyme and subjected to 2% agarose electrophoresis. An uninfected EB5 cell and a gfp-silenced EB5 cell (EB5-Mut1) were used as controls. The percentage of mutation was calculated using ImageJ. Abbreviations: bp, base pair; EB5, GFP-expressing B5 mouse embryonic stem cell line; GFP, green fluorescent protein; ssODN, single-stranded oligonucleotide DNA; TALEN, transcription activator-like effector nuclease.

Figure 4. — TALEN-mediated single-base change of gfp gene on genomic DNA. (A): Strategy of single-base modification in gfp gene. The ssODN template with a single base change from the wild-type sequence introduces a stop codon and a BfaI restriction enzyme digestion site. The second ssODN removes the stop codon and adds a new SacI restriction enzyme digestion site. (B): Fluorescent-activated cell sorting (FACS) analysis of fluorescence cell population 3 days after either transfection of ssODN-1 and eukaryotic expression plasmids encoding the TALEN pair (transfection; BII) or transfection of ssODN-1 followed by injection of TALEN proteins by P. aeruginosa (transfection-injection; BIII). As a control, an untreated EB5 cell is shown (BI). Percentages of the GFP-negative cells in the whole population are shown. (C): A 350-bp fragment encompassing the TALEN-targeting region was amplified by polymerase chain reaction (PCR) from GFP-negative EB5 cells that were FACS-sorted after either transfection of ssODN-1 and TALEN coding plasmids or ssODN-1 transfection followed by TALEN protein injection. The PCR products were subjected to 2% agarose electrophoresis with (+) or without (−) digestion by BfaI restriction enzyme. An uninfected EB5 cell was used as the control. M represents the DNA marker. The percentage of mutation was calculated using ImageJ. (D): Single cell cloning of EB5 with desired single-base change in the gfp gene. The gfp fragments were PCR amplified from 12 cell lines obtained by single cell cloning and subjected to 2% agarose electrophoresis after digestion by the BfaI restriction enzyme. Two desired cell lines, numbers 4 and 6, were obtained. (E): FACS analysis of fluorescence cell population 3 days after transfection of ssODN-2 and injection of TALEN proteins by P. aeruginosa. As a control, gfp-silenced EB5 cells (EB5-Mut1) were injected with the TALEN proteins only. The percentage of the GFP-positive cells in the whole population are shown. (F): After transfection of ssODN-2 and TALEN protein injection into EB5-Mut1, GFP-positive cells were FACS-sorted, and a 350-bp fragment encompassing the TALEN-targeting region was amplified by PCR. The PCR products were digested with (+) or without (−) the SacI restriction enzyme and subjected to 2% agarose electrophoresis. An uninfected EB5 cell and a gfp-silenced EB5 cell (EB5-Mut1) were used as controls. The percentage of mutation was calculated using ImageJ. Abbreviations: bp, base pair; EB5, GFP-expressing B5 mouse embryonic stem cell line; GFP, green fluorescent protein; ssODN, single-stranded oligonucleotide DNA; TALEN, transcription activator-like effector nuclease.