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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.


Transfection method for hard-to-transfect mammalian cells.(A) Illustration of the developed transfection procedure. Employed mammalian cells are capable of replicating plasmids by expressing the SV40 large T antigen. These cells are first electroporated with the plasmid (p10) which contains the mammalian antibiotic selection (e.g. neomycin) as well as the plasmid replication cassette (SV40 origin of replication). Next, electroporated mammalian cells are selected using 200 μg/ml neomycin (G 418) final concentrations. Selected cells are then electroporated with the second plasmid (p12). Following the second electroporation, cells are induced either by bradykinin or shear stress. (B) Green fluorescent cell percentages following the above described transfection procedure. Cells transfected with the method described in part (A) were either treated with Bradykinin or left untreated, as shown on the X-axis. Surface-adherent cells were stained with 2 μg/ml Hoechst 33342 and 0.5 μg/ml propidium iodide, fixed with 4% (v/v) paraformaldehyde, microscopy imaged, and counted in ImageJ. Determined green fluorescent cell percentages and cell viabilities are plotted on the left Y-axis alongside total cell numbers per images on the right Y-axis. Error bars represent ± SD, N = 3.
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f2: Transfection method for hard-to-transfect mammalian cells.(A) Illustration of the developed transfection procedure. Employed mammalian cells are capable of replicating plasmids by expressing the SV40 large T antigen. These cells are first electroporated with the plasmid (p10) which contains the mammalian antibiotic selection (e.g. neomycin) as well as the plasmid replication cassette (SV40 origin of replication). Next, electroporated mammalian cells are selected using 200 μg/ml neomycin (G 418) final concentrations. Selected cells are then electroporated with the second plasmid (p12). Following the second electroporation, cells are induced either by bradykinin or shear stress. (B) Green fluorescent cell percentages following the above described transfection procedure. Cells transfected with the method described in part (A) were either treated with Bradykinin or left untreated, as shown on the X-axis. Surface-adherent cells were stained with 2 μg/ml Hoechst 33342 and 0.5 μg/ml propidium iodide, fixed with 4% (v/v) paraformaldehyde, microscopy imaged, and counted in ImageJ. Determined green fluorescent cell percentages and cell viabilities are plotted on the left Y-axis alongside total cell numbers per images on the right Y-axis. Error bars represent ± SD, N = 3.

Mentions: Ultimately, to increase transfection efficiencies, the gene network was redesigned to reduce the number of encoding DNA plasmids from 3 to 2 (Fig. S2B). Furthermore, a mammalian antibiotic selection marker (neomycin), as well as a replication origin (Simian virus 40, SV40) for plasmid multiplication in mammalian cells was also included in one of the two plasmids (Fig. S2B). Simultaneous direct transfection of both plasmids did increase transfection efficiencies up to 30%. Consequently, an enrichment procedure was devised where the plasmid encoding the sensor and linker modules as well as an SV40 plasmid replication and an antibiotic (neomycin) resistance cassette was electroporated into cells, followed by antibiotic (neomycin) selection of plasmid-containing cells that were then further electroporated with the reporter plasmid (Fig. 2A). The SV40 replication system facilitated plasmid replication, thus plasmids with half-lives on the order of hours373839 were made available over the course of several days. Electroporating the improved network into HMEC-1 endothelial cells using the optimized procedure yielded greater than 60% green fluorescent cells when stimulated with Bradykinin (Fig. 2B).


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)

Transfection method for hard-to-transfect mammalian cells.(A) Illustration of the developed transfection procedure. Employed mammalian cells are capable of replicating plasmids by expressing the SV40 large T antigen. These cells are first electroporated with the plasmid (p10) which contains the mammalian antibiotic selection (e.g. neomycin) as well as the plasmid replication cassette (SV40 origin of replication). Next, electroporated mammalian cells are selected using 200 μg/ml neomycin (G 418) final concentrations. Selected cells are then electroporated with the second plasmid (p12). Following the second electroporation, cells are induced either by bradykinin or shear stress. (B) Green fluorescent cell percentages following the above described transfection procedure. Cells transfected with the method described in part (A) were either treated with Bradykinin or left untreated, as shown on the X-axis. Surface-adherent cells were stained with 2 μg/ml Hoechst 33342 and 0.5 μg/ml propidium iodide, fixed with 4% (v/v) paraformaldehyde, microscopy imaged, and counted in ImageJ. Determined green fluorescent cell percentages and cell viabilities are plotted on the left Y-axis alongside total cell numbers per images on the right Y-axis. Error bars represent ± SD, N = 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940741&req=5

f2: Transfection method for hard-to-transfect mammalian cells.(A) Illustration of the developed transfection procedure. Employed mammalian cells are capable of replicating plasmids by expressing the SV40 large T antigen. These cells are first electroporated with the plasmid (p10) which contains the mammalian antibiotic selection (e.g. neomycin) as well as the plasmid replication cassette (SV40 origin of replication). Next, electroporated mammalian cells are selected using 200 μg/ml neomycin (G 418) final concentrations. Selected cells are then electroporated with the second plasmid (p12). Following the second electroporation, cells are induced either by bradykinin or shear stress. (B) Green fluorescent cell percentages following the above described transfection procedure. Cells transfected with the method described in part (A) were either treated with Bradykinin or left untreated, as shown on the X-axis. Surface-adherent cells were stained with 2 μg/ml Hoechst 33342 and 0.5 μg/ml propidium iodide, fixed with 4% (v/v) paraformaldehyde, microscopy imaged, and counted in ImageJ. Determined green fluorescent cell percentages and cell viabilities are plotted on the left Y-axis alongside total cell numbers per images on the right Y-axis. Error bars represent ± SD, N = 3.
Mentions: Ultimately, to increase transfection efficiencies, the gene network was redesigned to reduce the number of encoding DNA plasmids from 3 to 2 (Fig. S2B). Furthermore, a mammalian antibiotic selection marker (neomycin), as well as a replication origin (Simian virus 40, SV40) for plasmid multiplication in mammalian cells was also included in one of the two plasmids (Fig. S2B). Simultaneous direct transfection of both plasmids did increase transfection efficiencies up to 30%. Consequently, an enrichment procedure was devised where the plasmid encoding the sensor and linker modules as well as an SV40 plasmid replication and an antibiotic (neomycin) resistance cassette was electroporated into cells, followed by antibiotic (neomycin) selection of plasmid-containing cells that were then further electroporated with the reporter plasmid (Fig. 2A). The SV40 replication system facilitated plasmid replication, thus plasmids with half-lives on the order of hours373839 were made available over the course of several days. Electroporating the improved network into HMEC-1 endothelial cells using the optimized procedure yielded greater than 60% green fluorescent cells when stimulated with Bradykinin (Fig. 2B).

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.