Limits...
Synthetic feedback control using an RNAi-based gene-regulatory device.

Bloom RJ, Winkler SM, Smolke CD - J Biol Eng (2015)

Bottom Line: Our work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device.The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands.The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA 94305 USA.

ABSTRACT

Background: Homeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response. The development of biological components that can respond to changes in the cellular environment and interface with endogenous molecules would enable more sophisticated genetic circuits and greatly advance our cellular engineering capabilities.

Results: Here we describe a platform that combines a ligand-responsive ribozyme switch and synthetic miRNA regulators to create an OFF genetic control device based on RNA interference (RNAi). We developed a mathematical model to highlight important design parameters in programming the quantitative performance of RNAi-based OFF control devices. By modifying the ribozyme switch integrated into the system, we demonstrated RNAi-based OFF control devices that respond to small molecule and protein ligands, including the oncogenic protein E2F1. We utilized the OFF control device platform to build a negative feedback control system that acts as a proportional controller and maintains target intracellular protein levels in response to increases in transcription rate.

Conclusions: Our work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device. The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands. Finally, the RNAi-based OFF switch can be used to implement a negative feedback control system, which maintains target protein levels around a set point level. The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

Protein-responsive RNAi-based OFF control devices. (a) Schematic overview of the genetic circuit used to characterize protein-responsive OFF control devices. The OFF control devices and GFP were stably integrated and expressed from identical expression cassettes as described in Figure 2a, except that the theophylline-responsive ribozyme was replaced with an MS2- or E2F1-responsive ribozyme switch. A plasmid encoding the expression of DsRed only (mock plasmid) or the protein ligand (MS2 or E2F1) was transfected into the cell lines to measure activity of the OFF control device. (b-c) GFP expression levels under the control of the MS2-responsive (b) or E2F1-responsive (c) OFF control device. No miR and miRNA only represent constructs with a protein-responsive ribozyme switch and no downstream miRNAs (negative control) and a construct with a non-cleaving ribozyme upstream of a GFP-targeting miRNA (positive control), respectively. Error bars represent ±1 standard deviation of at least three independent experiments. (d) Device dynamic ranges for the OFF control devices as a function of the ratio of cleavage rates (kcleave/kcleave-bound). Grey line: model; circle: theophylline-responsive device; square: MS2-responsive device. Device dynamic range is reported as the percent change in GFP expression in the absence and presence of theophylline. (e) Relative increase in miRNA levels after addition of protein ligand. Relative miRNA levels are reported normalized to the miRNA levels measured from identical cell lines in the absence of protein ligand. Error bars represent ±1 standard deviation of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4403951&req=5

Fig4: Protein-responsive RNAi-based OFF control devices. (a) Schematic overview of the genetic circuit used to characterize protein-responsive OFF control devices. The OFF control devices and GFP were stably integrated and expressed from identical expression cassettes as described in Figure 2a, except that the theophylline-responsive ribozyme was replaced with an MS2- or E2F1-responsive ribozyme switch. A plasmid encoding the expression of DsRed only (mock plasmid) or the protein ligand (MS2 or E2F1) was transfected into the cell lines to measure activity of the OFF control device. (b-c) GFP expression levels under the control of the MS2-responsive (b) or E2F1-responsive (c) OFF control device. No miR and miRNA only represent constructs with a protein-responsive ribozyme switch and no downstream miRNAs (negative control) and a construct with a non-cleaving ribozyme upstream of a GFP-targeting miRNA (positive control), respectively. Error bars represent ±1 standard deviation of at least three independent experiments. (d) Device dynamic ranges for the OFF control devices as a function of the ratio of cleavage rates (kcleave/kcleave-bound). Grey line: model; circle: theophylline-responsive device; square: MS2-responsive device. Device dynamic range is reported as the percent change in GFP expression in the absence and presence of theophylline. (e) Relative increase in miRNA levels after addition of protein ligand. Relative miRNA levels are reported normalized to the miRNA levels measured from identical cell lines in the absence of protein ligand. Error bars represent ±1 standard deviation of three independent experiments.

Mentions: The validated protein-responsive ribozyme switches were placed into the OFF control device platform to build an RNAi-based control system that decreased target gene expression levels as a function of increasing MS2 and E2F1 levels. Expression cassettes were constructed with the protein-responsive ribozymes placed 2500 bp upstream of a GFP-targeting miRNA (Figure 4a). A second expression cassette was constructed that encoded the expression of a GFP fluorescent reporter protein. Isogenic cell lines stably expressing both expression cassettes were generated from a Flp-In HEK293 cell line as previously described.Figure 4


Synthetic feedback control using an RNAi-based gene-regulatory device.

Bloom RJ, Winkler SM, Smolke CD - J Biol Eng (2015)

Protein-responsive RNAi-based OFF control devices. (a) Schematic overview of the genetic circuit used to characterize protein-responsive OFF control devices. The OFF control devices and GFP were stably integrated and expressed from identical expression cassettes as described in Figure 2a, except that the theophylline-responsive ribozyme was replaced with an MS2- or E2F1-responsive ribozyme switch. A plasmid encoding the expression of DsRed only (mock plasmid) or the protein ligand (MS2 or E2F1) was transfected into the cell lines to measure activity of the OFF control device. (b-c) GFP expression levels under the control of the MS2-responsive (b) or E2F1-responsive (c) OFF control device. No miR and miRNA only represent constructs with a protein-responsive ribozyme switch and no downstream miRNAs (negative control) and a construct with a non-cleaving ribozyme upstream of a GFP-targeting miRNA (positive control), respectively. Error bars represent ±1 standard deviation of at least three independent experiments. (d) Device dynamic ranges for the OFF control devices as a function of the ratio of cleavage rates (kcleave/kcleave-bound). Grey line: model; circle: theophylline-responsive device; square: MS2-responsive device. Device dynamic range is reported as the percent change in GFP expression in the absence and presence of theophylline. (e) Relative increase in miRNA levels after addition of protein ligand. Relative miRNA levels are reported normalized to the miRNA levels measured from identical cell lines in the absence of protein ligand. Error bars represent ±1 standard deviation of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403951&req=5

Fig4: Protein-responsive RNAi-based OFF control devices. (a) Schematic overview of the genetic circuit used to characterize protein-responsive OFF control devices. The OFF control devices and GFP were stably integrated and expressed from identical expression cassettes as described in Figure 2a, except that the theophylline-responsive ribozyme was replaced with an MS2- or E2F1-responsive ribozyme switch. A plasmid encoding the expression of DsRed only (mock plasmid) or the protein ligand (MS2 or E2F1) was transfected into the cell lines to measure activity of the OFF control device. (b-c) GFP expression levels under the control of the MS2-responsive (b) or E2F1-responsive (c) OFF control device. No miR and miRNA only represent constructs with a protein-responsive ribozyme switch and no downstream miRNAs (negative control) and a construct with a non-cleaving ribozyme upstream of a GFP-targeting miRNA (positive control), respectively. Error bars represent ±1 standard deviation of at least three independent experiments. (d) Device dynamic ranges for the OFF control devices as a function of the ratio of cleavage rates (kcleave/kcleave-bound). Grey line: model; circle: theophylline-responsive device; square: MS2-responsive device. Device dynamic range is reported as the percent change in GFP expression in the absence and presence of theophylline. (e) Relative increase in miRNA levels after addition of protein ligand. Relative miRNA levels are reported normalized to the miRNA levels measured from identical cell lines in the absence of protein ligand. Error bars represent ±1 standard deviation of three independent experiments.
Mentions: The validated protein-responsive ribozyme switches were placed into the OFF control device platform to build an RNAi-based control system that decreased target gene expression levels as a function of increasing MS2 and E2F1 levels. Expression cassettes were constructed with the protein-responsive ribozymes placed 2500 bp upstream of a GFP-targeting miRNA (Figure 4a). A second expression cassette was constructed that encoded the expression of a GFP fluorescent reporter protein. Isogenic cell lines stably expressing both expression cassettes were generated from a Flp-In HEK293 cell line as previously described.Figure 4

Bottom Line: Our work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device.The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands.The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA 94305 USA.

ABSTRACT

Background: Homeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response. The development of biological components that can respond to changes in the cellular environment and interface with endogenous molecules would enable more sophisticated genetic circuits and greatly advance our cellular engineering capabilities.

Results: Here we describe a platform that combines a ligand-responsive ribozyme switch and synthetic miRNA regulators to create an OFF genetic control device based on RNA interference (RNAi). We developed a mathematical model to highlight important design parameters in programming the quantitative performance of RNAi-based OFF control devices. By modifying the ribozyme switch integrated into the system, we demonstrated RNAi-based OFF control devices that respond to small molecule and protein ligands, including the oncogenic protein E2F1. We utilized the OFF control device platform to build a negative feedback control system that acts as a proportional controller and maintains target intracellular protein levels in response to increases in transcription rate.

Conclusions: Our work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device. The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands. Finally, the RNAi-based OFF switch can be used to implement a negative feedback control system, which maintains target protein levels around a set point level. The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.

No MeSH data available.


Related in: MedlinePlus