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Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3

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ABSTRACT

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp − 612 and − 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the − 612 and − 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

No MeSH data available.


In vivo imaging of liver sites where promoter luciferase constructs were introduced into a normal (NR) rat. Imaging was performed 24 hours after in vivo electroporation using a Xenogen in vivo Imager. Luciferase substrate was injected intraperitoneally prior to imaging. The regions of interest (ROI) are circled.
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f0005: In vivo imaging of liver sites where promoter luciferase constructs were introduced into a normal (NR) rat. Imaging was performed 24 hours after in vivo electroporation using a Xenogen in vivo Imager. Luciferase substrate was injected intraperitoneally prior to imaging. The regions of interest (ROI) are circled.

Mentions: Using our 5 mm hexagonal array electrode, we introduced rat LDL receptor promoter-luciferase reporter gene constructs and/or siRNAs, in duplicate, into each of three liver lobes in the same animal. This allows for direct comparisons. The area transfected is limited to that inside the hexagonal array. After 24 hours, the transfected areas were removed using a 5 mm cork borer. The precise location of the transfected regions is defined by six light dots due to electrode scaring on the liver surface. Luciferase activity is restricted to the 5 mm circle as demonstrated in Fig. 1.


Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3
In vivo imaging of liver sites where promoter luciferase constructs were introduced into a normal (NR) rat. Imaging was performed 24 hours after in vivo electroporation using a Xenogen in vivo Imager. Luciferase substrate was injected intraperitoneally prior to imaging. The regions of interest (ROI) are circled.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121208&req=5

f0005: In vivo imaging of liver sites where promoter luciferase constructs were introduced into a normal (NR) rat. Imaging was performed 24 hours after in vivo electroporation using a Xenogen in vivo Imager. Luciferase substrate was injected intraperitoneally prior to imaging. The regions of interest (ROI) are circled.
Mentions: Using our 5 mm hexagonal array electrode, we introduced rat LDL receptor promoter-luciferase reporter gene constructs and/or siRNAs, in duplicate, into each of three liver lobes in the same animal. This allows for direct comparisons. The area transfected is limited to that inside the hexagonal array. After 24 hours, the transfected areas were removed using a 5 mm cork borer. The precise location of the transfected regions is defined by six light dots due to electrode scaring on the liver surface. Luciferase activity is restricted to the 5 mm circle as demonstrated in Fig. 1.

View Article: PubMed Central - PubMed

ABSTRACT

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp − 612 and − 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the − 612 and − 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

No MeSH data available.