Limits...
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.


Design and computational validation of the in-house designed linear shear stress inducing flow chamber.(A) Geometry of the bespoke height-variance flow channel with shear stress map at the channel floor cell seeding area. Flow entered at the high height end of the channel and exited at the low height end. (B) Evolution of shear stress along the length of the channel floor, in the width-wise centre of the channel floor for the channel modelled in part A above. (C) Shear stress plotted across the width of the channel floor, in the length-wise centre of the channel floor, for the above modelled channel. (D) Geometry of the bespoke height-variance flow channel with reverse flow direction shear stress map at the channel floor. Flow entered at the low height end of the channel and exited at the high height end of the channel. (E) Shear stress along the length of the channel floor, in the width-wise centre of the channel floor, for the channel modelled in part D above. (F) Shear stress computed across the width of the channel floor, in the length-wise centre of the channel floor, for the channel illustrated in part D above. Flow rate was 10 ml/min in all plots and illustrated models. For shear stress vs. channel length and width plots at 5 ml/min and 20 ml/min see Fig. S6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940741&req=5

f3: Design and computational validation of the in-house designed linear shear stress inducing flow chamber.(A) Geometry of the bespoke height-variance flow channel with shear stress map at the channel floor cell seeding area. Flow entered at the high height end of the channel and exited at the low height end. (B) Evolution of shear stress along the length of the channel floor, in the width-wise centre of the channel floor for the channel modelled in part A above. (C) Shear stress plotted across the width of the channel floor, in the length-wise centre of the channel floor, for the above modelled channel. (D) Geometry of the bespoke height-variance flow channel with reverse flow direction shear stress map at the channel floor. Flow entered at the low height end of the channel and exited at the high height end of the channel. (E) Shear stress along the length of the channel floor, in the width-wise centre of the channel floor, for the channel modelled in part D above. (F) Shear stress computed across the width of the channel floor, in the length-wise centre of the channel floor, for the channel illustrated in part D above. Flow rate was 10 ml/min in all plots and illustrated models. For shear stress vs. channel length and width plots at 5 ml/min and 20 ml/min see Fig. S6.

Mentions: In order to effectively apply a graded shear stress response in gene network-encoding cells, a flow chamber with variable height was designed. First, the 3D CAD model of the flow chamber with 0.5 cm width, 5 cm length and height varying along the chamber length described by a square root function, was generated in SolidWorks (Fig. 3A,D). The resultant shear stress on the bottom (cell-seeded) surface of the chamber, computed in SolidWorks’ Flow Simulation Package, increased linearly along the chamber length while shear stress was uniform across the chamber width (Fig. 3B,C,E,F). The chamber was developed and computationally validated for applying wall shear stress in the 0 to 10 Pa range, which can be decreased by lowering the flow rate (Fig. S6). Thus, this flow device facilitates studies in the entire shear stress range of human arteries, including shear stress below 1 Pa in atherosclerosis prone arterial regions and normal veins, between 1 Pa and 7 Pa in normal arteries, and between 7 Pa and 10 Pa in advanced plaques, cardiac valves and stents43. To obtain the physical flow channel with this rather complex geometry, two separate parts were used: a glass slide on which cells were seeded and a bottomless flow channel. The bottomless flow channel was obtained by casting polydimethylsiloxane (PDMS) on metal mold parts. Complementary equipment for cell seeding, clamping and microscopy visualization were also designed in SolidWorks and manufactured (Fig. S7). The purpose-built PDMS well was mounted atop of the glass slide to facilitate the seeding of cells on the glass slide. After cell seeding, the PDMS well part could be replaced with the bottomless flow channel to expose cells to shear stress. The complementary clamping and microscopy visualization equipment consisted of two metal plates in between which the bottomless flow channel is mounted on top of the glass slide, and the two metal plates were held together by screws. Both metal plates contained an opening in the channel area to allow light to pass through the equipment for visualization of cells under microscopes. The bottom metal plate was designed to fit into the stage of conventional microscopes.


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 and computational validation of the in-house designed linear shear stress inducing flow chamber.(A) Geometry of the bespoke height-variance flow channel with shear stress map at the channel floor cell seeding area. Flow entered at the high height end of the channel and exited at the low height end. (B) Evolution of shear stress along the length of the channel floor, in the width-wise centre of the channel floor for the channel modelled in part A above. (C) Shear stress plotted across the width of the channel floor, in the length-wise centre of the channel floor, for the above modelled channel. (D) Geometry of the bespoke height-variance flow channel with reverse flow direction shear stress map at the channel floor. Flow entered at the low height end of the channel and exited at the high height end of the channel. (E) Shear stress along the length of the channel floor, in the width-wise centre of the channel floor, for the channel modelled in part D above. (F) Shear stress computed across the width of the channel floor, in the length-wise centre of the channel floor, for the channel illustrated in part D above. Flow rate was 10 ml/min in all plots and illustrated models. For shear stress vs. channel length and width plots at 5 ml/min and 20 ml/min see Fig. S6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Design and computational validation of the in-house designed linear shear stress inducing flow chamber.(A) Geometry of the bespoke height-variance flow channel with shear stress map at the channel floor cell seeding area. Flow entered at the high height end of the channel and exited at the low height end. (B) Evolution of shear stress along the length of the channel floor, in the width-wise centre of the channel floor for the channel modelled in part A above. (C) Shear stress plotted across the width of the channel floor, in the length-wise centre of the channel floor, for the above modelled channel. (D) Geometry of the bespoke height-variance flow channel with reverse flow direction shear stress map at the channel floor. Flow entered at the low height end of the channel and exited at the high height end of the channel. (E) Shear stress along the length of the channel floor, in the width-wise centre of the channel floor, for the channel modelled in part D above. (F) Shear stress computed across the width of the channel floor, in the length-wise centre of the channel floor, for the channel illustrated in part D above. Flow rate was 10 ml/min in all plots and illustrated models. For shear stress vs. channel length and width plots at 5 ml/min and 20 ml/min see Fig. S6.
Mentions: In order to effectively apply a graded shear stress response in gene network-encoding cells, a flow chamber with variable height was designed. First, the 3D CAD model of the flow chamber with 0.5 cm width, 5 cm length and height varying along the chamber length described by a square root function, was generated in SolidWorks (Fig. 3A,D). The resultant shear stress on the bottom (cell-seeded) surface of the chamber, computed in SolidWorks’ Flow Simulation Package, increased linearly along the chamber length while shear stress was uniform across the chamber width (Fig. 3B,C,E,F). The chamber was developed and computationally validated for applying wall shear stress in the 0 to 10 Pa range, which can be decreased by lowering the flow rate (Fig. S6). Thus, this flow device facilitates studies in the entire shear stress range of human arteries, including shear stress below 1 Pa in atherosclerosis prone arterial regions and normal veins, between 1 Pa and 7 Pa in normal arteries, and between 7 Pa and 10 Pa in advanced plaques, cardiac valves and stents43. To obtain the physical flow channel with this rather complex geometry, two separate parts were used: a glass slide on which cells were seeded and a bottomless flow channel. The bottomless flow channel was obtained by casting polydimethylsiloxane (PDMS) on metal mold parts. Complementary equipment for cell seeding, clamping and microscopy visualization were also designed in SolidWorks and manufactured (Fig. S7). The purpose-built PDMS well was mounted atop of the glass slide to facilitate the seeding of cells on the glass slide. After cell seeding, the PDMS well part could be replaced with the bottomless flow channel to expose cells to shear stress. The complementary clamping and microscopy visualization equipment consisted of two metal plates in between which the bottomless flow channel is mounted on top of the glass slide, and the two metal plates were held together by screws. Both metal plates contained an opening in the channel area to allow light to pass through the equipment for visualization of cells under microscopes. The bottom metal plate was designed to fit into the stage of conventional microscopes.

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.