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Design of a zinc finger protein binding a sequence upstream of the A20 gene.

Wei Y, Ying D, Hou C, Cui X, Zhu C - BMC Biotechnol. (2008)

Bottom Line: Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods.The ZFP was designed successfully and exhibited biological activity.It is feasible to design specific zinc finger proteins by bioinformatics methods.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Key Laboratory of Biomechanics and Tissue Engineering of Chongqing Municipality, Department of Anatomy, Third Military Medical University, Chongqing, 400038, China. yongwei70@gmail.com

ABSTRACT

Background: Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.

Results: In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.

Conclusion: It is feasible to design specific zinc finger proteins by bioinformatics methods.

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Expression of green fluorescent protein in COS-7 cells transfected with recombinant plasmids pIRES2-EGFP/ZFP-flag seen through the fluorescence microscope. A: Expression 24 h after transfection (100×). B: Expression 36 h after transfection (200×). The result of the pIRES2-EGFP control group was omitted.
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Figure 3: Expression of green fluorescent protein in COS-7 cells transfected with recombinant plasmids pIRES2-EGFP/ZFP-flag seen through the fluorescence microscope. A: Expression 24 h after transfection (100×). B: Expression 36 h after transfection (200×). The result of the pIRES2-EGFP control group was omitted.

Mentions: In the pIRES2-EGFP/ZFP-flag transfection group, cells emitting green fluorescence were observed under the microscope 12 h after transfection; 24 h after transfection there were significantly more of these cells, up to dozens in each visual field (100×), mostly emitting bright green fluorescence. The fluorescence intensity reached a peak 48 h after transfection, and the cells were found to form masses or pairs, with no significant differences between the nuclear and cytoplasmic fluorescence intensities. In order to determine the transfection efficiency, the cells were counted in 10 random visual fields (100×) 36 h after transfection. Of the 519 cells counted, 227 emitted green fluorescence (43.7%). In the pIRES2-EGFP control group, green fluorescence emitting cells were first observed 8 h after transfection. Moreover, the number of cells emitting green fluorescence and the fluorescence intensity were higher in the pIRES2-EGFP control group than in the pIRES2-EGFP/ZFP-flag transfection group at various times of cell culture. In the control group, no green fluorescence was observed (Fig. 3).


Design of a zinc finger protein binding a sequence upstream of the A20 gene.

Wei Y, Ying D, Hou C, Cui X, Zhu C - BMC Biotechnol. (2008)

Expression of green fluorescent protein in COS-7 cells transfected with recombinant plasmids pIRES2-EGFP/ZFP-flag seen through the fluorescence microscope. A: Expression 24 h after transfection (100×). B: Expression 36 h after transfection (200×). The result of the pIRES2-EGFP control group was omitted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2278136&req=5

Figure 3: Expression of green fluorescent protein in COS-7 cells transfected with recombinant plasmids pIRES2-EGFP/ZFP-flag seen through the fluorescence microscope. A: Expression 24 h after transfection (100×). B: Expression 36 h after transfection (200×). The result of the pIRES2-EGFP control group was omitted.
Mentions: In the pIRES2-EGFP/ZFP-flag transfection group, cells emitting green fluorescence were observed under the microscope 12 h after transfection; 24 h after transfection there were significantly more of these cells, up to dozens in each visual field (100×), mostly emitting bright green fluorescence. The fluorescence intensity reached a peak 48 h after transfection, and the cells were found to form masses or pairs, with no significant differences between the nuclear and cytoplasmic fluorescence intensities. In order to determine the transfection efficiency, the cells were counted in 10 random visual fields (100×) 36 h after transfection. Of the 519 cells counted, 227 emitted green fluorescence (43.7%). In the pIRES2-EGFP control group, green fluorescence emitting cells were first observed 8 h after transfection. Moreover, the number of cells emitting green fluorescence and the fluorescence intensity were higher in the pIRES2-EGFP control group than in the pIRES2-EGFP/ZFP-flag transfection group at various times of cell culture. In the control group, no green fluorescence was observed (Fig. 3).

Bottom Line: Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods.The ZFP was designed successfully and exhibited biological activity.It is feasible to design specific zinc finger proteins by bioinformatics methods.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Key Laboratory of Biomechanics and Tissue Engineering of Chongqing Municipality, Department of Anatomy, Third Military Medical University, Chongqing, 400038, China. yongwei70@gmail.com

ABSTRACT

Background: Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.

Results: In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.

Conclusion: It is feasible to design specific zinc finger proteins by bioinformatics methods.

Show MeSH
Related in: MedlinePlus