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Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

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Related in: MedlinePlus

Expression of the ATF in the ECV304 cells. Immunofluorescence was performed, and the resulting cells were observed under a laser scanning confocal microscope. (a) ECV304 cells transfected with pcDNA3.1-NLS-AZP-p65-flag. (b) ECV304 cells transfected with pcDNA3.1-AZP-p65-flag. (c) ECV304 cells transfected with the control plasmids. The white arrows pointed the expressed proteins which were located in the nuclei.
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fig4: Expression of the ATF in the ECV304 cells. Immunofluorescence was performed, and the resulting cells were observed under a laser scanning confocal microscope. (a) ECV304 cells transfected with pcDNA3.1-NLS-AZP-p65-flag. (b) ECV304 cells transfected with pcDNA3.1-AZP-p65-flag. (c) ECV304 cells transfected with the control plasmids. The white arrows pointed the expressed proteins which were located in the nuclei.

Mentions: The flag tag on the ATF vectors was used for protein expression detection. Immunofluorescence showed that the ECV304 cells, which were transfected with ATF vectors, were Cy3-positive, whereas the control cells were not (Figure 4). This indicated that after vector transfection, the ATF could be normally expressed in ECV304 cells.


Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Expression of the ATF in the ECV304 cells. Immunofluorescence was performed, and the resulting cells were observed under a laser scanning confocal microscope. (a) ECV304 cells transfected with pcDNA3.1-NLS-AZP-p65-flag. (b) ECV304 cells transfected with pcDNA3.1-AZP-p65-flag. (c) ECV304 cells transfected with the control plasmids. The white arrows pointed the expressed proteins which were located in the nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2913762&req=5

fig4: Expression of the ATF in the ECV304 cells. Immunofluorescence was performed, and the resulting cells were observed under a laser scanning confocal microscope. (a) ECV304 cells transfected with pcDNA3.1-NLS-AZP-p65-flag. (b) ECV304 cells transfected with pcDNA3.1-AZP-p65-flag. (c) ECV304 cells transfected with the control plasmids. The white arrows pointed the expressed proteins which were located in the nuclei.
Mentions: The flag tag on the ATF vectors was used for protein expression detection. Immunofluorescence showed that the ECV304 cells, which were transfected with ATF vectors, were Cy3-positive, whereas the control cells were not (Figure 4). This indicated that after vector transfection, the ATF could be normally expressed in ECV304 cells.

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

Show MeSH
Related in: MedlinePlus