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Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs.

Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S, Qin L, Chen X - Cell Regen (Lond) (2013)

Bottom Line: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research.Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency.This new technique may advance the clinical application of TALEN technology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.

Results: In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.

Conclusions: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.

No MeSH data available.


Related in: MedlinePlus

Purification of TAT-ZFNs. (A) Schematic diagram of the TAT-ZFN construct. (B) SDS-PAGE analysis of TAT-ZFNs purified under native conditions. (C) Western blot analysis of expressed TAT-ZFNs using an anti-His antibody. (D) In vitro activity testing of TAT-ZFNs purified under native conditions. The negative control is indicated by “-”. The recognition sequence of Hpy188III is located within the ZFN recognition sequence; therefore, Hpy188III was used as the positive control (+).
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Fig1: Purification of TAT-ZFNs. (A) Schematic diagram of the TAT-ZFN construct. (B) SDS-PAGE analysis of TAT-ZFNs purified under native conditions. (C) Western blot analysis of expressed TAT-ZFNs using an anti-His antibody. (D) In vitro activity testing of TAT-ZFNs purified under native conditions. The negative control is indicated by “-”. The recognition sequence of Hpy188III is located within the ZFN recognition sequence; therefore, Hpy188III was used as the positive control (+).

Mentions: The structure of the TAT-ZFN construct is shown in Figure 1A. Soluble proteins were purified as described in Methods. TAT-ZFN purification was greatly limited by its low protein expression level and low binding affinity; thus, only a small amount of TAT-ZFN could be obtained (Figure 1B). Because the purification tag could be detected by anti-His antibodies (Figure 1C), we speculated that the His tag was likely to be hidden by the tertiary structure of the native protein. Nevertheless, TAT-ZFNs showed specific nuclease activity in vitro (Figure 1D).Figure 1


Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs.

Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S, Qin L, Chen X - Cell Regen (Lond) (2013)

Purification of TAT-ZFNs. (A) Schematic diagram of the TAT-ZFN construct. (B) SDS-PAGE analysis of TAT-ZFNs purified under native conditions. (C) Western blot analysis of expressed TAT-ZFNs using an anti-His antibody. (D) In vitro activity testing of TAT-ZFNs purified under native conditions. The negative control is indicated by “-”. The recognition sequence of Hpy188III is located within the ZFN recognition sequence; therefore, Hpy188III was used as the positive control (+).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230761&req=5

Fig1: Purification of TAT-ZFNs. (A) Schematic diagram of the TAT-ZFN construct. (B) SDS-PAGE analysis of TAT-ZFNs purified under native conditions. (C) Western blot analysis of expressed TAT-ZFNs using an anti-His antibody. (D) In vitro activity testing of TAT-ZFNs purified under native conditions. The negative control is indicated by “-”. The recognition sequence of Hpy188III is located within the ZFN recognition sequence; therefore, Hpy188III was used as the positive control (+).
Mentions: The structure of the TAT-ZFN construct is shown in Figure 1A. Soluble proteins were purified as described in Methods. TAT-ZFN purification was greatly limited by its low protein expression level and low binding affinity; thus, only a small amount of TAT-ZFN could be obtained (Figure 1B). Because the purification tag could be detected by anti-His antibodies (Figure 1C), we speculated that the His tag was likely to be hidden by the tertiary structure of the native protein. Nevertheless, TAT-ZFNs showed specific nuclease activity in vitro (Figure 1D).Figure 1

Bottom Line: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research.Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency.This new technique may advance the clinical application of TALEN technology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.

Results: In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.

Conclusions: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.

No MeSH data available.


Related in: MedlinePlus