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The food additive vanillic acid controls transgene expression in mammalian cells and mice.

Gitzinger M, Kemmer C, Fluri DA, El-Baba MD, Weber W, Fussenegger M - Nucleic Acids Res. (2011)

Bottom Line: Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells.When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism.As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.

ABSTRACT
Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

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Vanillic acid-controlled SEAP expression in mice. (A) CHO-VAC12 cells were microencapsulated in alginate-poly-(l-lysine)-alginate beads and implanted intraperitoneally into female OF1 mice (4 × 106 cells per mouse). The implanted mice received different concentrations of vanillic acid twice daily. Seventy-two hours after implantation, the level of SEAP in the serum of the mice was determined. Data represent mean ± SEM of 8 mice per treatment group. (B) SEAP expression profiles of the microencapsulated CHO-VAC12 implant batch were cultivated in vitro for 72 h at different vanillic acid concentrations. (C and D) Extracts of wild-type mouse organs were assessed for their vanillic acid content based on their ability to induce the (C) VACOFF or (D) VACON systems. Vanillic acid-spiked organs were used as positive control. All samples were compared to the effect of 250 μM vanillic acid to show the fully induced state of the systems. All extracts were added to CHO-K1 cells transiently transfected with either the VACON or the VACOFF systems and SEAP expression was assessed after a cultivation period of 48 h.
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gkr1251-F5: Vanillic acid-controlled SEAP expression in mice. (A) CHO-VAC12 cells were microencapsulated in alginate-poly-(l-lysine)-alginate beads and implanted intraperitoneally into female OF1 mice (4 × 106 cells per mouse). The implanted mice received different concentrations of vanillic acid twice daily. Seventy-two hours after implantation, the level of SEAP in the serum of the mice was determined. Data represent mean ± SEM of 8 mice per treatment group. (B) SEAP expression profiles of the microencapsulated CHO-VAC12 implant batch were cultivated in vitro for 72 h at different vanillic acid concentrations. (C and D) Extracts of wild-type mouse organs were assessed for their vanillic acid content based on their ability to induce the (C) VACOFF or (D) VACON systems. Vanillic acid-spiked organs were used as positive control. All samples were compared to the effect of 250 μM vanillic acid to show the fully induced state of the systems. All extracts were added to CHO-K1 cells transiently transfected with either the VACON or the VACOFF systems and SEAP expression was assessed after a cultivation period of 48 h.

Mentions: For subsequent applications in functional genomics research or future gene- and cell-based therapies, it is essential that state-of-the-art gene regulation systems are functional within entire organisms. To validate the vanillic acid-controlled gene regulation system in vivo, we implanted microencapsulated CHO-VAC12 intraperitoneally into mice. The treated mice were given a dose of vanillic acid within the range of 0–500 mg/kg twice daily. SEAP levels quantified in the blood of treated animals 72 h after implantation showed vanillic acid-dependent dose–response characteristics comparable to the control experiment using the same batch of microencapsulated CHO-VAC12 cells exposed to vanillic acid in an in vitro setting (Figure 5A and B). The serum SEAP levels of control mice, encapsulated with constitutively expressing CHO-SEAP18, were unresponsive to vanillic acid treatment of a twice-daily dose of 500 mg/kg and thus showed similar expression levels as untreated mice containing CHO-VAC12 implants (0 mg/kg vanillic acid: 15.37 ± 1.57 U/l; 500 mg/kg vanillic acid: 16.61 ± 1.33 U/l). Since vanillic acid is a standard food additive it may in principle be present in the animal and interfere with VACOFF-based fine-tuning of transgenes. We have therefore analysed whether liver, kidney, muscle and lung tissue extracts of mice kept on a standard diet could interfere with CHO-K1 cultures engineered for VACOFF- and VACON-controlled SEAP expression. Unlike positive controls consisting of organ extracts spiked with vanillic acid, none of the organ extracts produced from wild-type mice kept on a standard diet showed any interference with the VACON or VACOFF systems (Figure 5C and D).Figure 5.


The food additive vanillic acid controls transgene expression in mammalian cells and mice.

Gitzinger M, Kemmer C, Fluri DA, El-Baba MD, Weber W, Fussenegger M - Nucleic Acids Res. (2011)

Vanillic acid-controlled SEAP expression in mice. (A) CHO-VAC12 cells were microencapsulated in alginate-poly-(l-lysine)-alginate beads and implanted intraperitoneally into female OF1 mice (4 × 106 cells per mouse). The implanted mice received different concentrations of vanillic acid twice daily. Seventy-two hours after implantation, the level of SEAP in the serum of the mice was determined. Data represent mean ± SEM of 8 mice per treatment group. (B) SEAP expression profiles of the microencapsulated CHO-VAC12 implant batch were cultivated in vitro for 72 h at different vanillic acid concentrations. (C and D) Extracts of wild-type mouse organs were assessed for their vanillic acid content based on their ability to induce the (C) VACOFF or (D) VACON systems. Vanillic acid-spiked organs were used as positive control. All samples were compared to the effect of 250 μM vanillic acid to show the fully induced state of the systems. All extracts were added to CHO-K1 cells transiently transfected with either the VACON or the VACOFF systems and SEAP expression was assessed after a cultivation period of 48 h.
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Related In: Results  -  Collection

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gkr1251-F5: Vanillic acid-controlled SEAP expression in mice. (A) CHO-VAC12 cells were microencapsulated in alginate-poly-(l-lysine)-alginate beads and implanted intraperitoneally into female OF1 mice (4 × 106 cells per mouse). The implanted mice received different concentrations of vanillic acid twice daily. Seventy-two hours after implantation, the level of SEAP in the serum of the mice was determined. Data represent mean ± SEM of 8 mice per treatment group. (B) SEAP expression profiles of the microencapsulated CHO-VAC12 implant batch were cultivated in vitro for 72 h at different vanillic acid concentrations. (C and D) Extracts of wild-type mouse organs were assessed for their vanillic acid content based on their ability to induce the (C) VACOFF or (D) VACON systems. Vanillic acid-spiked organs were used as positive control. All samples were compared to the effect of 250 μM vanillic acid to show the fully induced state of the systems. All extracts were added to CHO-K1 cells transiently transfected with either the VACON or the VACOFF systems and SEAP expression was assessed after a cultivation period of 48 h.
Mentions: For subsequent applications in functional genomics research or future gene- and cell-based therapies, it is essential that state-of-the-art gene regulation systems are functional within entire organisms. To validate the vanillic acid-controlled gene regulation system in vivo, we implanted microencapsulated CHO-VAC12 intraperitoneally into mice. The treated mice were given a dose of vanillic acid within the range of 0–500 mg/kg twice daily. SEAP levels quantified in the blood of treated animals 72 h after implantation showed vanillic acid-dependent dose–response characteristics comparable to the control experiment using the same batch of microencapsulated CHO-VAC12 cells exposed to vanillic acid in an in vitro setting (Figure 5A and B). The serum SEAP levels of control mice, encapsulated with constitutively expressing CHO-SEAP18, were unresponsive to vanillic acid treatment of a twice-daily dose of 500 mg/kg and thus showed similar expression levels as untreated mice containing CHO-VAC12 implants (0 mg/kg vanillic acid: 15.37 ± 1.57 U/l; 500 mg/kg vanillic acid: 16.61 ± 1.33 U/l). Since vanillic acid is a standard food additive it may in principle be present in the animal and interfere with VACOFF-based fine-tuning of transgenes. We have therefore analysed whether liver, kidney, muscle and lung tissue extracts of mice kept on a standard diet could interfere with CHO-K1 cultures engineered for VACOFF- and VACON-controlled SEAP expression. Unlike positive controls consisting of organ extracts spiked with vanillic acid, none of the organ extracts produced from wild-type mice kept on a standard diet showed any interference with the VACON or VACOFF systems (Figure 5C and D).Figure 5.

Bottom Line: Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells.When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism.As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.

ABSTRACT
Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

Show MeSH
Related in: MedlinePlus