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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

Western blot analysis of lysate from the treated HeLa cells using an anti-FLAG antibody. HeLa cells were incubated with TALEN for 1 hour at 37°C and subsequently washed three times with 1 mg/ml heparin-PBS to remove proteins bound to the cell surface before lysis.
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Fig4: Western blot analysis of lysate from the treated HeLa cells using an anti-FLAG antibody. HeLa cells were incubated with TALEN for 1 hour at 37°C and subsequently washed three times with 1 mg/ml heparin-PBS to remove proteins bound to the cell surface before lysis.

Mentions: To evaluate the ability of the purified proteins to penetrate cells, HeLa cells were treated with 1.5 μM TALEN proteins for 1 hour at 37°C and subsequently washed with 1 mg/ml heparin to remove proteins bound to the cell surface. Immunoblotting of the treated HeLa lysate showed that TALEN itself could not penetrate the cells; however, the 11-amino acid TAT was able to deliver the 110 kD TALEN into the cells (Figure 4).Figure 4


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)

Western blot analysis of lysate from the treated HeLa cells using an anti-FLAG antibody. HeLa cells were incubated with TALEN for 1 hour at 37°C and subsequently washed three times with 1 mg/ml heparin-PBS to remove proteins bound to the cell surface before lysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Western blot analysis of lysate from the treated HeLa cells using an anti-FLAG antibody. HeLa cells were incubated with TALEN for 1 hour at 37°C and subsequently washed three times with 1 mg/ml heparin-PBS to remove proteins bound to the cell surface before lysis.
Mentions: To evaluate the ability of the purified proteins to penetrate cells, HeLa cells were treated with 1.5 μM TALEN proteins for 1 hour at 37°C and subsequently washed with 1 mg/ml heparin to remove proteins bound to the cell surface. Immunoblotting of the treated HeLa lysate showed that TALEN itself could not penetrate the cells; however, the 11-amino acid TAT was able to deliver the 110 kD TALEN into the cells (Figure 4).Figure 4

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