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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.


Gene expression modulation by mechanical stimuli.(A) The KLF2-eGFP fusion protein of the gene network up-regulates downstream eNOS and THB genes. HMEC-1 cells were either transfected with the gene network following the method describe in Fig. 2, or left untransfected. All cells were exposed to a shear stress of 2 Pa for 24 hours. Next, eNOS and THB mRNA levels were quantified relative to untransfected static (i.e., no flow) controls. Two-way ANOVA showed a statistical increase (p < 0.0001, N = 3) in the mRNA levels of both eNOS and THB in gene network transfected flow exposed cells compared to flow exposed cells without the gene network. (B) KLF2-eGFP expression modulation by shear stress in HMEC-1 cells. Green fluorescent cell percentages vary logarithmically as a function of shear stress (N = 3 flow chambers). All error bars represent ± SD.
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f5: Gene expression modulation by mechanical stimuli.(A) The KLF2-eGFP fusion protein of the gene network up-regulates downstream eNOS and THB genes. HMEC-1 cells were either transfected with the gene network following the method describe in Fig. 2, or left untransfected. All cells were exposed to a shear stress of 2 Pa for 24 hours. Next, eNOS and THB mRNA levels were quantified relative to untransfected static (i.e., no flow) controls. Two-way ANOVA showed a statistical increase (p < 0.0001, N = 3) in the mRNA levels of both eNOS and THB in gene network transfected flow exposed cells compared to flow exposed cells without the gene network. (B) KLF2-eGFP expression modulation by shear stress in HMEC-1 cells. Green fluorescent cell percentages vary logarithmically as a function of shear stress (N = 3 flow chambers). All error bars represent ± SD.

Mentions: We subsequently modified the network to modulate the expression of a known mechanosensitive transcription factor. We chose Krueppel like factor 2 (KLF2) because it is an important mechanotransduction transcription factor that regulates greater than 50% of mechanosensitive genes in endothelial cells535455. Towards this end, the existing reporter plasmid (p12) was modified by replacing the eGFP gene with a KLF2-eGFP fusion gene (Fig. S9). The gene network (encoded on p10 and p14) was electroporated into HMEC-1 endothelial cells and functionality of the network was first assessed by exposing the cells to increasing concentrations of Bradykinin, which activated expression of KLF2-eGFP in a dose-dependent manner (Fig. S10). To validate that the KLF2 gene within the synthetic network behaves similarly to endogenously expressed KLF2, particularly with the addition of the fused GFP tag, HMEC-1 cells were either electroporated with the network or left untransfected and the mRNA levels of two genes (endothelial nitric oxide, eNOS, and thrombomodulin, THB) known to be upregulated by KLF2545657 were examined after 24 hours of flow relative to the mRNA levels in untransfected static (i.e., no flow) controls (Fig. 5A). A two-way analysis of variance (ANOVA) was used to assess the influence of the network and gene type on gene expression within the different cell populations. Presence of the gene network caused a statistically significant increase in expression of eNOS and THB compared to untransfected cells (p < 0.0001, N = 3). In addition, THB mRNA levels were statistically higher (p < 0.001, N = 3) than eNOS mRNA levels. No statistically significant interaction was found between the network and gene type variables (p = 0.086). To examine whether we could monitor the activity of KLF2 over increasing levels of shear stress, the cells were seeded into our custom microfluidic device and exposed to the same flow regimen described above. Similar to our original synthetic network, results showed increasing levels of GFP with increasing shear stress that was best described by a logarithmic function, demonstrating that we could directly modulate KLF2 expression by shear stress (Fig. 5B).


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)

Gene expression modulation by mechanical stimuli.(A) The KLF2-eGFP fusion protein of the gene network up-regulates downstream eNOS and THB genes. HMEC-1 cells were either transfected with the gene network following the method describe in Fig. 2, or left untransfected. All cells were exposed to a shear stress of 2 Pa for 24 hours. Next, eNOS and THB mRNA levels were quantified relative to untransfected static (i.e., no flow) controls. Two-way ANOVA showed a statistical increase (p < 0.0001, N = 3) in the mRNA levels of both eNOS and THB in gene network transfected flow exposed cells compared to flow exposed cells without the gene network. (B) KLF2-eGFP expression modulation by shear stress in HMEC-1 cells. Green fluorescent cell percentages vary logarithmically as a function of shear stress (N = 3 flow chambers). All error bars represent ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Gene expression modulation by mechanical stimuli.(A) The KLF2-eGFP fusion protein of the gene network up-regulates downstream eNOS and THB genes. HMEC-1 cells were either transfected with the gene network following the method describe in Fig. 2, or left untransfected. All cells were exposed to a shear stress of 2 Pa for 24 hours. Next, eNOS and THB mRNA levels were quantified relative to untransfected static (i.e., no flow) controls. Two-way ANOVA showed a statistical increase (p < 0.0001, N = 3) in the mRNA levels of both eNOS and THB in gene network transfected flow exposed cells compared to flow exposed cells without the gene network. (B) KLF2-eGFP expression modulation by shear stress in HMEC-1 cells. Green fluorescent cell percentages vary logarithmically as a function of shear stress (N = 3 flow chambers). All error bars represent ± SD.
Mentions: We subsequently modified the network to modulate the expression of a known mechanosensitive transcription factor. We chose Krueppel like factor 2 (KLF2) because it is an important mechanotransduction transcription factor that regulates greater than 50% of mechanosensitive genes in endothelial cells535455. Towards this end, the existing reporter plasmid (p12) was modified by replacing the eGFP gene with a KLF2-eGFP fusion gene (Fig. S9). The gene network (encoded on p10 and p14) was electroporated into HMEC-1 endothelial cells and functionality of the network was first assessed by exposing the cells to increasing concentrations of Bradykinin, which activated expression of KLF2-eGFP in a dose-dependent manner (Fig. S10). To validate that the KLF2 gene within the synthetic network behaves similarly to endogenously expressed KLF2, particularly with the addition of the fused GFP tag, HMEC-1 cells were either electroporated with the network or left untransfected and the mRNA levels of two genes (endothelial nitric oxide, eNOS, and thrombomodulin, THB) known to be upregulated by KLF2545657 were examined after 24 hours of flow relative to the mRNA levels in untransfected static (i.e., no flow) controls (Fig. 5A). A two-way analysis of variance (ANOVA) was used to assess the influence of the network and gene type on gene expression within the different cell populations. Presence of the gene network caused a statistically significant increase in expression of eNOS and THB compared to untransfected cells (p < 0.0001, N = 3). In addition, THB mRNA levels were statistically higher (p < 0.001, N = 3) than eNOS mRNA levels. No statistically significant interaction was found between the network and gene type variables (p = 0.086). To examine whether we could monitor the activity of KLF2 over increasing levels of shear stress, the cells were seeded into our custom microfluidic device and exposed to the same flow regimen described above. Similar to our original synthetic network, results showed increasing levels of GFP with increasing shear stress that was best described by a logarithmic function, demonstrating that we could directly modulate KLF2 expression by shear stress (Fig. 5B).

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.