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Development of a synthetic gene network to modulate gene expression by mechanical forces.

Kis Z, Rodin T, Zafar A, Lai Z, Freke G, Fleck O, Del Rio Hernandez A, Towhidi L, Pedrigi RM, Homma T, Krams R - Sci Rep (2016)

Bottom Line: We call this new approach mechanosyngenetics.To insert the gene network into a high proportion of cells, a hybrid transfection procedure was developed that involves electroporation, plasmids replication in mammalian cells, mammalian antibiotic selection, a second electroporation and gene network activation.This procedure takes 1 week and yielded over 60% of cells with a functional gene network.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Imperial College London, Exhibition Road, London, SW7 2AZ, United Kingdom.

ABSTRACT
The majority of (mammalian) cells in our body are sensitive to mechanical forces, but little work has been done to develop assays to monitor mechanosensor activity. Furthermore, it is currently impossible to use mechanosensor activity to drive gene expression. To address these needs, we developed the first mammalian mechanosensitive synthetic gene network to monitor endothelial cell shear stress levels and directly modulate expression of an atheroprotective transcription factor by shear stress. The technique is highly modular, easily scalable and allows graded control of gene expression by mechanical stimuli in hard-to-transfect mammalian cells. We call this new approach mechanosyngenetics. To insert the gene network into a high proportion of cells, a hybrid transfection procedure was developed that involves electroporation, plasmids replication in mammalian cells, mammalian antibiotic selection, a second electroporation and gene network activation. This procedure takes 1 week and yielded over 60% of cells with a functional gene network. To test gene network functionality, we developed a flow setup that exposes cells to linearly increasing shear stress along the length of the flow channel floor. Activation of the gene network varied logarithmically as a function of shear stress magnitude.

No MeSH data available.


Related in: MedlinePlus

Design of the mechanosensitive synthetic gene network and its validation in HeLa and endothelial EA.hy926 and HMEC-1 cells.(A) Illustration of salient network components. Shear stress induction of the G-PCR causes Arrestin binding to the GPCR tail domain (TL), leading to cleavage of the TEV cleavage site linker by TEV. Released tTA promotes transcription of the eGFP reporter gene. (B) The gene network is functional in HeLa cells. Superimposed green fluorescent and phase contrast microscopy images of HeLa cells transfected with the gene network under static (i.e. no flow) conditions. HeLa cells were electroporated with the gene network and were either treated with 2 μM final bradykinin concentrations or were left untreated. Bradykinin treatment yielded ≈10% green fluorescent HeLa cells. N = 3, S.D.≈10%, 10X magnification. (C) The gene network is functional in endothelial EA.hy926 and HMEC-1 cells. Superimposed green fluorescent and phase contrast microscopy images of EA.hy926 and HMEC-1 endothelial cells transfected with the gene network. Cells were electroporated with the gene network and subjected either to 2 Pa shear stress for 24 hours, or to 2 μM bradykinin under static conditions, or left untreated under static conditions. Bradykinin treatment and flow exposure yielded ≈10% green fluorescent EA.hy926 and HMEC-1 cells. N = 3, S.D.≈10%, 10X magnification.
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f1: Design of the mechanosensitive synthetic gene network and its validation in HeLa and endothelial EA.hy926 and HMEC-1 cells.(A) Illustration of salient network components. Shear stress induction of the G-PCR causes Arrestin binding to the GPCR tail domain (TL), leading to cleavage of the TEV cleavage site linker by TEV. Released tTA promotes transcription of the eGFP reporter gene. (B) The gene network is functional in HeLa cells. Superimposed green fluorescent and phase contrast microscopy images of HeLa cells transfected with the gene network under static (i.e. no flow) conditions. HeLa cells were electroporated with the gene network and were either treated with 2 μM final bradykinin concentrations or were left untreated. Bradykinin treatment yielded ≈10% green fluorescent HeLa cells. N = 3, S.D.≈10%, 10X magnification. (C) The gene network is functional in endothelial EA.hy926 and HMEC-1 cells. Superimposed green fluorescent and phase contrast microscopy images of EA.hy926 and HMEC-1 endothelial cells transfected with the gene network. Cells were electroporated with the gene network and subjected either to 2 Pa shear stress for 24 hours, or to 2 μM bradykinin under static conditions, or left untreated under static conditions. Bradykinin treatment and flow exposure yielded ≈10% green fluorescent EA.hy926 and HMEC-1 cells. N = 3, S.D.≈10%, 10X magnification.

Mentions: We designed our synthetic gene network to assay the mechanosensitive G-PCR activity by extensive modification of an existing assay24 (Fig. S2). Our mechanosensitive network consists of a sensor module, a reporter module, and a linker module that signals between the sensor and reporter (Fig. 1A and supplemental methods). The sensor module is composed of the Bradykinin B2 G-PCR23 linked to the tetracycline-controlled transcriptional trans-activator (tTA), with the linker consisting of a 7 amino acid long Tobacco etch virus (TEV) protease cleavage site. The reporter module contains eGFP under the control of a tTA dependent promoter. β-Arrestin2 signalling protein fused to the TEV protease enzyme constitutes the linker module. Upon activation of the G-PCR by shear stress or by an extracellular ligand, β-Arrestin2 binds to the cytoplasmic domain of the G-PCR leading to cleavage of the linker between tTA and the G-PCR by the TEV protease. Consequently, the released tTA enters the cell nucleus and promotes transcription from the reporter module. The gene network can be controlled at the shear stress sensor level by activating and inhibiting the Bradykinin B2 G-PCR, using bradykinin252627 and HOE 14028293031 ligands, respectively. Additionally, the gene network can be inhibited at the transcription level using doxycycline3233 (Fig. S3). After the gene network was constructed at the DNA level, it was inserted into mammalian cells to validate its functionality (Fig. 1B,C). It was also confirmed that the GFP is expressed in response to shear stress only when all components of the gene network are present, see Fig. S4 for details.


Development of a synthetic gene network to modulate gene expression by mechanical forces.

Kis Z, Rodin T, Zafar A, Lai Z, Freke G, Fleck O, Del Rio Hernandez A, Towhidi L, Pedrigi RM, Homma T, Krams R - Sci Rep (2016)

Design of the mechanosensitive synthetic gene network and its validation in HeLa and endothelial EA.hy926 and HMEC-1 cells.(A) Illustration of salient network components. Shear stress induction of the G-PCR causes Arrestin binding to the GPCR tail domain (TL), leading to cleavage of the TEV cleavage site linker by TEV. Released tTA promotes transcription of the eGFP reporter gene. (B) The gene network is functional in HeLa cells. Superimposed green fluorescent and phase contrast microscopy images of HeLa cells transfected with the gene network under static (i.e. no flow) conditions. HeLa cells were electroporated with the gene network and were either treated with 2 μM final bradykinin concentrations or were left untreated. Bradykinin treatment yielded ≈10% green fluorescent HeLa cells. N = 3, S.D.≈10%, 10X magnification. (C) The gene network is functional in endothelial EA.hy926 and HMEC-1 cells. Superimposed green fluorescent and phase contrast microscopy images of EA.hy926 and HMEC-1 endothelial cells transfected with the gene network. Cells were electroporated with the gene network and subjected either to 2 Pa shear stress for 24 hours, or to 2 μM bradykinin under static conditions, or left untreated under static conditions. Bradykinin treatment and flow exposure yielded ≈10% green fluorescent EA.hy926 and HMEC-1 cells. N = 3, S.D.≈10%, 10X magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940741&req=5

f1: Design of the mechanosensitive synthetic gene network and its validation in HeLa and endothelial EA.hy926 and HMEC-1 cells.(A) Illustration of salient network components. Shear stress induction of the G-PCR causes Arrestin binding to the GPCR tail domain (TL), leading to cleavage of the TEV cleavage site linker by TEV. Released tTA promotes transcription of the eGFP reporter gene. (B) The gene network is functional in HeLa cells. Superimposed green fluorescent and phase contrast microscopy images of HeLa cells transfected with the gene network under static (i.e. no flow) conditions. HeLa cells were electroporated with the gene network and were either treated with 2 μM final bradykinin concentrations or were left untreated. Bradykinin treatment yielded ≈10% green fluorescent HeLa cells. N = 3, S.D.≈10%, 10X magnification. (C) The gene network is functional in endothelial EA.hy926 and HMEC-1 cells. Superimposed green fluorescent and phase contrast microscopy images of EA.hy926 and HMEC-1 endothelial cells transfected with the gene network. Cells were electroporated with the gene network and subjected either to 2 Pa shear stress for 24 hours, or to 2 μM bradykinin under static conditions, or left untreated under static conditions. Bradykinin treatment and flow exposure yielded ≈10% green fluorescent EA.hy926 and HMEC-1 cells. N = 3, S.D.≈10%, 10X magnification.
Mentions: We designed our synthetic gene network to assay the mechanosensitive G-PCR activity by extensive modification of an existing assay24 (Fig. S2). Our mechanosensitive network consists of a sensor module, a reporter module, and a linker module that signals between the sensor and reporter (Fig. 1A and supplemental methods). The sensor module is composed of the Bradykinin B2 G-PCR23 linked to the tetracycline-controlled transcriptional trans-activator (tTA), with the linker consisting of a 7 amino acid long Tobacco etch virus (TEV) protease cleavage site. The reporter module contains eGFP under the control of a tTA dependent promoter. β-Arrestin2 signalling protein fused to the TEV protease enzyme constitutes the linker module. Upon activation of the G-PCR by shear stress or by an extracellular ligand, β-Arrestin2 binds to the cytoplasmic domain of the G-PCR leading to cleavage of the linker between tTA and the G-PCR by the TEV protease. Consequently, the released tTA enters the cell nucleus and promotes transcription from the reporter module. The gene network can be controlled at the shear stress sensor level by activating and inhibiting the Bradykinin B2 G-PCR, using bradykinin252627 and HOE 14028293031 ligands, respectively. Additionally, the gene network can be inhibited at the transcription level using doxycycline3233 (Fig. S3). After the gene network was constructed at the DNA level, it was inserted into mammalian cells to validate its functionality (Fig. 1B,C). It was also confirmed that the GFP is expressed in response to shear stress only when all components of the gene network are present, see Fig. S4 for details.

Bottom Line: We call this new approach mechanosyngenetics.To insert the gene network into a high proportion of cells, a hybrid transfection procedure was developed that involves electroporation, plasmids replication in mammalian cells, mammalian antibiotic selection, a second electroporation and gene network activation.This procedure takes 1 week and yielded over 60% of cells with a functional gene network.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Imperial College London, Exhibition Road, London, SW7 2AZ, United Kingdom.

ABSTRACT
The majority of (mammalian) cells in our body are sensitive to mechanical forces, but little work has been done to develop assays to monitor mechanosensor activity. Furthermore, it is currently impossible to use mechanosensor activity to drive gene expression. To address these needs, we developed the first mammalian mechanosensitive synthetic gene network to monitor endothelial cell shear stress levels and directly modulate expression of an atheroprotective transcription factor by shear stress. The technique is highly modular, easily scalable and allows graded control of gene expression by mechanical stimuli in hard-to-transfect mammalian cells. We call this new approach mechanosyngenetics. To insert the gene network into a high proportion of cells, a hybrid transfection procedure was developed that involves electroporation, plasmids replication in mammalian cells, mammalian antibiotic selection, a second electroporation and gene network activation. This procedure takes 1 week and yielded over 60% of cells with a functional gene network. To test gene network functionality, we developed a flow setup that exposes cells to linearly increasing shear stress along the length of the flow channel floor. Activation of the gene network varied logarithmically as a function of shear stress magnitude.

No MeSH data available.


Related in: MedlinePlus