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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.


The gene network senses graded levels of shear stress.(A) Graded gene network activation by shear stress. HMEC-1 cells were transfected with the procedure described in Fig. 2 and seeded in the flow channel shown in Fig. 3 for activation by linearly increasing shear stress. Following the flow experiment, cells were stained with Hoechst 33342 and propidium iodide, fixed with paraformaldehyde, and microscopy imaged and counted in ImageJ. Plotted GFP expressing HMEC-1 cell percentages versus shear stress shows a logarithmic relationship (N = 7 flow chambers). Error bars represent ± SD. (B) Pilot flow experiment with reversed flow direction (high shear to low shear). Cells were treated as described in part A with the exception of the flow direction being connected in the opposite direction in the flow channel (high shear to low shear). GFP expressing HMEC-1 cell percentages vary logarithmically in function of shear stress (N = 1 flow channel, 4 sets of images). (C) Representative fluorescent images of HMEC-1 endothelial cells electroporated with the synthetic network and exposed to different levels of shear stress (based on position within the microfluidic device), as described in part A. Green fluorescence (GFP) indicates gene network activity and blue fluorescence (Hoechst) shows the total number of cells.
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f4: The gene network senses graded levels of shear stress.(A) Graded gene network activation by shear stress. HMEC-1 cells were transfected with the procedure described in Fig. 2 and seeded in the flow channel shown in Fig. 3 for activation by linearly increasing shear stress. Following the flow experiment, cells were stained with Hoechst 33342 and propidium iodide, fixed with paraformaldehyde, and microscopy imaged and counted in ImageJ. Plotted GFP expressing HMEC-1 cell percentages versus shear stress shows a logarithmic relationship (N = 7 flow chambers). Error bars represent ± SD. (B) Pilot flow experiment with reversed flow direction (high shear to low shear). Cells were treated as described in part A with the exception of the flow direction being connected in the opposite direction in the flow channel (high shear to low shear). GFP expressing HMEC-1 cell percentages vary logarithmically in function of shear stress (N = 1 flow channel, 4 sets of images). (C) Representative fluorescent images of HMEC-1 endothelial cells electroporated with the synthetic network and exposed to different levels of shear stress (based on position within the microfluidic device), as described in part A. Green fluorescence (GFP) indicates gene network activity and blue fluorescence (Hoechst) shows the total number of cells.

Mentions: To explore the sensitivity of our gene network to graded levels of shear stress, HMEC-1 endothelial cells were electroporated with the gene network (encoded on p10 and p12, Table S1), using the above described transfection procedure (Fig. 2) and were seeded in the custom flow chamber for exposure to the shear stress range of 0 to 5 Pa for 24 hours. Cells were then fixed and the entire length of the chamber was imaged using fluorescence microscopy (Fig. 4C). Results showed that increasing shear stress caused accumulative G-PCR activation that was best fit by a logarithmic function (Fig. 4A). As a consequence, the G-PCR displayed a higher sensitivity to discriminate changes in shear stress at physiological values of 0 to 2 Pa. To eliminate the possibility that release of signaling molecules by cells in the upstream (low shear) region of the chamber might affect the activation of the gene network in cells of the downstream (high shear) region, we reversed the flow direction in the device. Results showed a logarithmic relationship between increasing shear stress and G-PCR activation (Fig. 4B), similar to the case of direct flow experiments. Analysis of covariance (ANCOVA) did not show differences between the two logarithmic graphs, proving that the increase in shear stress, and not paracrine signaling, caused the increase in G-PCR activation. To the best of our knowledge, this is the first synthetic network capable of detecting a graded response to shear stress in hard-to-transfect endothelial cells.


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)

The gene network senses graded levels of shear stress.(A) Graded gene network activation by shear stress. HMEC-1 cells were transfected with the procedure described in Fig. 2 and seeded in the flow channel shown in Fig. 3 for activation by linearly increasing shear stress. Following the flow experiment, cells were stained with Hoechst 33342 and propidium iodide, fixed with paraformaldehyde, and microscopy imaged and counted in ImageJ. Plotted GFP expressing HMEC-1 cell percentages versus shear stress shows a logarithmic relationship (N = 7 flow chambers). Error bars represent ± SD. (B) Pilot flow experiment with reversed flow direction (high shear to low shear). Cells were treated as described in part A with the exception of the flow direction being connected in the opposite direction in the flow channel (high shear to low shear). GFP expressing HMEC-1 cell percentages vary logarithmically in function of shear stress (N = 1 flow channel, 4 sets of images). (C) Representative fluorescent images of HMEC-1 endothelial cells electroporated with the synthetic network and exposed to different levels of shear stress (based on position within the microfluidic device), as described in part A. Green fluorescence (GFP) indicates gene network activity and blue fluorescence (Hoechst) shows the total number of cells.
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f4: The gene network senses graded levels of shear stress.(A) Graded gene network activation by shear stress. HMEC-1 cells were transfected with the procedure described in Fig. 2 and seeded in the flow channel shown in Fig. 3 for activation by linearly increasing shear stress. Following the flow experiment, cells were stained with Hoechst 33342 and propidium iodide, fixed with paraformaldehyde, and microscopy imaged and counted in ImageJ. Plotted GFP expressing HMEC-1 cell percentages versus shear stress shows a logarithmic relationship (N = 7 flow chambers). Error bars represent ± SD. (B) Pilot flow experiment with reversed flow direction (high shear to low shear). Cells were treated as described in part A with the exception of the flow direction being connected in the opposite direction in the flow channel (high shear to low shear). GFP expressing HMEC-1 cell percentages vary logarithmically in function of shear stress (N = 1 flow channel, 4 sets of images). (C) Representative fluorescent images of HMEC-1 endothelial cells electroporated with the synthetic network and exposed to different levels of shear stress (based on position within the microfluidic device), as described in part A. Green fluorescence (GFP) indicates gene network activity and blue fluorescence (Hoechst) shows the total number of cells.
Mentions: To explore the sensitivity of our gene network to graded levels of shear stress, HMEC-1 endothelial cells were electroporated with the gene network (encoded on p10 and p12, Table S1), using the above described transfection procedure (Fig. 2) and were seeded in the custom flow chamber for exposure to the shear stress range of 0 to 5 Pa for 24 hours. Cells were then fixed and the entire length of the chamber was imaged using fluorescence microscopy (Fig. 4C). Results showed that increasing shear stress caused accumulative G-PCR activation that was best fit by a logarithmic function (Fig. 4A). As a consequence, the G-PCR displayed a higher sensitivity to discriminate changes in shear stress at physiological values of 0 to 2 Pa. To eliminate the possibility that release of signaling molecules by cells in the upstream (low shear) region of the chamber might affect the activation of the gene network in cells of the downstream (high shear) region, we reversed the flow direction in the device. Results showed a logarithmic relationship between increasing shear stress and G-PCR activation (Fig. 4B), similar to the case of direct flow experiments. Analysis of covariance (ANCOVA) did not show differences between the two logarithmic graphs, proving that the increase in shear stress, and not paracrine signaling, caused the increase in G-PCR activation. To the best of our knowledge, this is the first synthetic network capable of detecting a graded response to shear stress in hard-to-transfect endothelial cells.

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.