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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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Validation of vanillic acid-responsive promoter variants containing different numbers of VanO operator modules. Vectors encoding SEAP expression driven by a vanillic acid-responsive promoter harbouring monomeric (pMG262), dimeric (pMG252), trimeric (pMG263) or tetrameric (pMG264) operator modules were co-transfected with pMG250 (PSV40-VanA1-pA) into (A) CHO-K1, (B) HEK-293 and (C) BHK-21 cells and SEAP production was scored after cultivation for 48 h in the presence and absence 250 µM vanillic acid.
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gkr1251-F2: Validation of vanillic acid-responsive promoter variants containing different numbers of VanO operator modules. Vectors encoding SEAP expression driven by a vanillic acid-responsive promoter harbouring monomeric (pMG262), dimeric (pMG252), trimeric (pMG263) or tetrameric (pMG264) operator modules were co-transfected with pMG250 (PSV40-VanA1-pA) into (A) CHO-K1, (B) HEK-293 and (C) BHK-21 cells and SEAP production was scored after cultivation for 48 h in the presence and absence 250 µM vanillic acid.

Mentions: Altering the number of operator modules in front of an inducible minimal promoter impacts the regulation performance regarding (i) maximal expression levels, as an increasing number of operator sites can recruit more transactivators and (ii) basal expression of the system's repressed state, as more transactivators have to be released from an increasing number of operator sites (27,55). To evaluate the optimal number of VanO-operator sites for VACOFF-controlled gene regulation, we constructed VanA1-responsive promoter variants harbouring either one (pMG262, P1VanO1-SEAP-pA; P1VanO1, NruI-VanO-0bp-PhCMVmin), two (pMG252, P1VanO2-SEAP-pA; P1VanO2, NruI-VanO-Eco47III-VanO-0bp-PhCMVmin), three (pMG263, P1VanO3-SEAP-pA; P1VanO3, NruI-VanO-Eco47III-VanO-6bp-VanO-0bp-PhCMVmin) or four (pMG264, P1VanO4-SEAP-pA; P1VanO4, NruI-VanO-Eco47III-VanO-6bp-VanO-Eco47III-VanO-0bp-PhCMVmin) operators immediately 5′ of the minimal promoter. Co-transfection of corresponding SEAP expression vectors harbouring the different promoter variants with pMG250 (PSV40-VanA1-pA) into CHO-K1, HEK-293 and BHK-21 cells and scoring of SEAP levels 48 h after cultivation in the presence and absence of 250 μM vanillic acid revealed their transcriptional performances. In all cell lines, it was observed that an increasing number of operator modules resulted in higher maximum expression levels but also in higher basal expression of the repressed status. The dimeric promoter configuration (pMG252, P1VanO2-SEAP-pA) resulted in the best regulation performance with a 72-fold induction factor in CHO-KI cells and 23-fold in HEK-293 cells, while the trimeric promoter set-up showed the best performance in BHK-21 cells with a ON/OFF control factor of 11 (Figure 2A–C).Figure 2.


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)

Validation of vanillic acid-responsive promoter variants containing different numbers of VanO operator modules. Vectors encoding SEAP expression driven by a vanillic acid-responsive promoter harbouring monomeric (pMG262), dimeric (pMG252), trimeric (pMG263) or tetrameric (pMG264) operator modules were co-transfected with pMG250 (PSV40-VanA1-pA) into (A) CHO-K1, (B) HEK-293 and (C) BHK-21 cells and SEAP production was scored after cultivation for 48 h in the presence and absence 250 µM vanillic acid.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3300003&req=5

gkr1251-F2: Validation of vanillic acid-responsive promoter variants containing different numbers of VanO operator modules. Vectors encoding SEAP expression driven by a vanillic acid-responsive promoter harbouring monomeric (pMG262), dimeric (pMG252), trimeric (pMG263) or tetrameric (pMG264) operator modules were co-transfected with pMG250 (PSV40-VanA1-pA) into (A) CHO-K1, (B) HEK-293 and (C) BHK-21 cells and SEAP production was scored after cultivation for 48 h in the presence and absence 250 µM vanillic acid.
Mentions: Altering the number of operator modules in front of an inducible minimal promoter impacts the regulation performance regarding (i) maximal expression levels, as an increasing number of operator sites can recruit more transactivators and (ii) basal expression of the system's repressed state, as more transactivators have to be released from an increasing number of operator sites (27,55). To evaluate the optimal number of VanO-operator sites for VACOFF-controlled gene regulation, we constructed VanA1-responsive promoter variants harbouring either one (pMG262, P1VanO1-SEAP-pA; P1VanO1, NruI-VanO-0bp-PhCMVmin), two (pMG252, P1VanO2-SEAP-pA; P1VanO2, NruI-VanO-Eco47III-VanO-0bp-PhCMVmin), three (pMG263, P1VanO3-SEAP-pA; P1VanO3, NruI-VanO-Eco47III-VanO-6bp-VanO-0bp-PhCMVmin) or four (pMG264, P1VanO4-SEAP-pA; P1VanO4, NruI-VanO-Eco47III-VanO-6bp-VanO-Eco47III-VanO-0bp-PhCMVmin) operators immediately 5′ of the minimal promoter. Co-transfection of corresponding SEAP expression vectors harbouring the different promoter variants with pMG250 (PSV40-VanA1-pA) into CHO-K1, HEK-293 and BHK-21 cells and scoring of SEAP levels 48 h after cultivation in the presence and absence of 250 μM vanillic acid revealed their transcriptional performances. In all cell lines, it was observed that an increasing number of operator modules resulted in higher maximum expression levels but also in higher basal expression of the repressed status. The dimeric promoter configuration (pMG252, P1VanO2-SEAP-pA) resulted in the best regulation performance with a 72-fold induction factor in CHO-KI cells and 23-fold in HEK-293 cells, while the trimeric promoter set-up showed the best performance in BHK-21 cells with a ON/OFF control factor of 11 (Figure 2A–C).Figure 2.

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