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A vanillic acid inducible expression system for Trypanosoma brucei.

Sunter JD - Mol. Biochem. Parasitol. (2016)

Bottom Line: Reverse genetics in Trypanosoma brucei is dependent on the tetracycline inducible system for the precise control over the expression of both genes and dsRNA.Another independent inducible system for trypanosomes would enable the control of the activities of two different genes in the same cell, providing greater experimental sophistication.Here, I describe the development of the vanillic acid based inducible expression system for T. brucei, which operates independently of, and can be used in parallel with the tetracycline inducible system.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, UK. Electronic address: jack.sunter@path.ox.ac.uk.

No MeSH data available.


Vanillic acid and doxycycline inducible systems can be used simultaneously and vanillic acid inducible system supports RNAi knockdown. A) Images of 927 pJ1173 pJ1271 pJ1225 cells induced for 24 h with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline. Scale bar is 10 μm. B) Western blot of PFR2 depletion after induction of PFR2 RNAi with vanillic acid using the L8C4 antibody. 2 × 106 cell equivalents were loaded per lane with the Ponceau staining as a loading control.
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fig0010: Vanillic acid and doxycycline inducible systems can be used simultaneously and vanillic acid inducible system supports RNAi knockdown. A) Images of 927 pJ1173 pJ1271 pJ1225 cells induced for 24 h with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline. Scale bar is 10 μm. B) Western blot of PFR2 depletion after induction of PFR2 RNAi with vanillic acid using the L8C4 antibody. 2 × 106 cell equivalents were loaded per lane with the Ponceau staining as a loading control.

Mentions: To show that the two induction systems could operate simultaneously, a modified version of pDEX777 called pJ1225 with eGFP replaced with dTomFP was integrated into the 927 pJ1173 pJ1271 cell line. This cell line was then treated with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline for 24 h; the cells were then fixed and imaged (Fig. 2A). When either vanillic acid or doxycycline was present the cells only expressed eGFP or dTomFP respectively, and when both were present the cells were able to express both eGFP and dTomFP. This demonstrates that the cells are able to support expression from both the vanillic acid and tetracycline inducible systems simultaneously and that the two systems are independent of each other. To show that the vanillic acid inducible system was capable of causing protein depletion by RNAi, a hairpin RNAi construct designed against PFR2 (Tb927.8.4970) was cloned into plasmid pJ1271 to give pJ1285, which was then integrated into the 927 pJ1173 cell line. PFR2 RNAi was induced by the addition of vanillic acid and depletion of PFR2 was followed by western blotting (Fig. 2B).


A vanillic acid inducible expression system for Trypanosoma brucei.

Sunter JD - Mol. Biochem. Parasitol. (2016)

Vanillic acid and doxycycline inducible systems can be used simultaneously and vanillic acid inducible system supports RNAi knockdown. A) Images of 927 pJ1173 pJ1271 pJ1225 cells induced for 24 h with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline. Scale bar is 10 μm. B) Western blot of PFR2 depletion after induction of PFR2 RNAi with vanillic acid using the L8C4 antibody. 2 × 106 cell equivalents were loaded per lane with the Ponceau staining as a loading control.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4920640&req=5

fig0010: Vanillic acid and doxycycline inducible systems can be used simultaneously and vanillic acid inducible system supports RNAi knockdown. A) Images of 927 pJ1173 pJ1271 pJ1225 cells induced for 24 h with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline. Scale bar is 10 μm. B) Western blot of PFR2 depletion after induction of PFR2 RNAi with vanillic acid using the L8C4 antibody. 2 × 106 cell equivalents were loaded per lane with the Ponceau staining as a loading control.
Mentions: To show that the two induction systems could operate simultaneously, a modified version of pDEX777 called pJ1225 with eGFP replaced with dTomFP was integrated into the 927 pJ1173 pJ1271 cell line. This cell line was then treated with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline for 24 h; the cells were then fixed and imaged (Fig. 2A). When either vanillic acid or doxycycline was present the cells only expressed eGFP or dTomFP respectively, and when both were present the cells were able to express both eGFP and dTomFP. This demonstrates that the cells are able to support expression from both the vanillic acid and tetracycline inducible systems simultaneously and that the two systems are independent of each other. To show that the vanillic acid inducible system was capable of causing protein depletion by RNAi, a hairpin RNAi construct designed against PFR2 (Tb927.8.4970) was cloned into plasmid pJ1271 to give pJ1285, which was then integrated into the 927 pJ1173 cell line. PFR2 RNAi was induced by the addition of vanillic acid and depletion of PFR2 was followed by western blotting (Fig. 2B).

Bottom Line: Reverse genetics in Trypanosoma brucei is dependent on the tetracycline inducible system for the precise control over the expression of both genes and dsRNA.Another independent inducible system for trypanosomes would enable the control of the activities of two different genes in the same cell, providing greater experimental sophistication.Here, I describe the development of the vanillic acid based inducible expression system for T. brucei, which operates independently of, and can be used in parallel with the tetracycline inducible system.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, UK. Electronic address: jack.sunter@path.ox.ac.uk.

No MeSH data available.