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

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Figure 5. — Type III secretion system (T3SS)-mediated injection of TALEN proteins into human ESCs and iPSCs. (A): Percentage of reduction of GFP-positive LT2e-H9CAGGFP cells after infection by the PAK-J∆8/pExoS54-FLAG-TALEN1 and PAK-J∆8/pExoS54-FLAG-TALEN2 (1:1 ratio) at the indicated MOI for 3 hours. The data were compared with those of untreated controls using a two-sample t test. ∗∗, p < .001. Error bars represent SD of triplicate assays. (B): Fluorescence intensity of LT2e-H9CAGGFP cells transfected by eukaryotic expression plasmids encoding gfp-targeting TALEN pair or infected by a 1:1 mixture of PAK-J∆8/pExoS54-FLAG-TALEN1 and PAK-J∆8/pExoS54-FLAG-TALEN2. Cells were analyzed by flow cytometry 3 days after treatment. The data were compared with those from untreated control using a two-sample t test. ∗∗∗, p < .0001. Error bars represent SD of triplicate assays. (C): A representative GFP-negative LT2e-H9CAGGFP cell cluster after bacterial delivery of gfp-targeting TALEN protein pair, observed under fluorescence microscope. (D): Schematic representation of TALEN binding sites on HPRT1 gene. The left and right TALEN binding sequences are shown in green and blue, respectively, and the spacer sequence is shown in red. (E): Sequence changes in the HPRT1 target site among iPSCs surviving the 6-thio-guanime (6TG) selection after P. aeruginosa-mediated TALEN delivery. (F): Strategy of single-base modification in the HPRT1 gene. The 72-base-pair-long ssODN-3 introduces a stop codon in the HPRT open reading frame and eliminates an XhoI restriction enzyme recognition site. (G): A polymerase chain reaction fragment of the HPRT1 gene was amplified from iPSCs surviving 6TG selection after gene modification by the ssODN-3 and P. aeruginosa-mediated TALEN delivery. The DNA fragments were digested (+) or undigested (−) with XhoI before being subjected to electrophoresis on a 0.8% agarose gel. Untreated iPSCs and iPSCs injected with the TALEN but without the ssODN-3 template were used as negative controls. The percentage of mutation was calculated using ImageJ. (H): Sequence changes in the HPRT1 target site among iPSCs surviving 6TG selection after P. aeruginosa-mediated TALEN delivery and ssODN-3 transfection. Abbreviations: ESCs, embryonic stem cells; ExoS54, exotoxin S N-terminal 54 amino acids fused with Cre recombinase; GFP, green fluorescent protein; hESCs, human embryonic stem cells; iPSCs, induced pluripotent stem cell; MOI, multiplicity of infection; TALEN, transcription activator-like effector nuclease; WT, wild type.

Figure 5. — Type III secretion system (T3SS)-mediated injection of TALEN proteins into human ESCs and iPSCs. (A): Percentage of reduction of GFP-positive LT2e-H9CAGGFP cells after infection by the PAK-J∆8/pExoS54-FLAG-TALEN1 and PAK-J∆8/pExoS54-FLAG-TALEN2 (1:1 ratio) at the indicated MOI for 3 hours. The data were compared with those of untreated controls using a two-sample t test. ∗∗, p < .001. Error bars represent SD of triplicate assays. (B): Fluorescence intensity of LT2e-H9CAGGFP cells transfected by eukaryotic expression plasmids encoding gfp-targeting TALEN pair or infected by a 1:1 mixture of PAK-J∆8/pExoS54-FLAG-TALEN1 and PAK-J∆8/pExoS54-FLAG-TALEN2. Cells were analyzed by flow cytometry 3 days after treatment. The data were compared with those from untreated control using a two-sample t test. ∗∗∗, p < .0001. Error bars represent SD of triplicate assays. (C): A representative GFP-negative LT2e-H9CAGGFP cell cluster after bacterial delivery of gfp-targeting TALEN protein pair, observed under fluorescence microscope. (D): Schematic representation of TALEN binding sites on HPRT1 gene. The left and right TALEN binding sequences are shown in green and blue, respectively, and the spacer sequence is shown in red. (E): Sequence changes in the HPRT1 target site among iPSCs surviving the 6-thio-guanime (6TG) selection after P. aeruginosa-mediated TALEN delivery. (F): Strategy of single-base modification in the HPRT1 gene. The 72-base-pair-long ssODN-3 introduces a stop codon in the HPRT open reading frame and eliminates an XhoI restriction enzyme recognition site. (G): A polymerase chain reaction fragment of the HPRT1 gene was amplified from iPSCs surviving 6TG selection after gene modification by the ssODN-3 and P. aeruginosa-mediated TALEN delivery. The DNA fragments were digested (+) or undigested (−) with XhoI before being subjected to electrophoresis on a 0.8% agarose gel. Untreated iPSCs and iPSCs injected with the TALEN but without the ssODN-3 template were used as negative controls. The percentage of mutation was calculated using ImageJ. (H): Sequence changes in the HPRT1 target site among iPSCs surviving 6TG selection after P. aeruginosa-mediated TALEN delivery and ssODN-3 transfection. Abbreviations: ESCs, embryonic stem cells; ExoS54, exotoxin S N-terminal 54 amino acids fused with Cre recombinase; GFP, green fluorescent protein; hESCs, human embryonic stem cells; iPSCs, induced pluripotent stem cell; MOI, multiplicity of infection; TALEN, transcription activator-like effector nuclease; WT, wild type.