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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2008 Apr 1;1779(11):712–719. doi: 10.1016/j.bbagrm.2008.03.007

Figure 1. ARE and ΔARE reporter constructs and their expression in mammalian cells.

Figure 1

A)Schematic representation of the ARE and ΔARE constructs. The full length mouse TNF-α 3’UTR was added downstream of the rabbit β-globin gene (ARE reporter). The AU-rich elements in the TNF-α 3’UTR were deleted to generate the ΔARE reporter. Both the ARE and ΔARE reporters were constructed under the control of the cytomegalovirus (CMV) or Tet promoters. The quantitative RT-PCR detection system spans intron 2. si20-targeting site and AU-rich elements (ARE) are localized in the 3’UTR of the constructs. Bovine growth hormone poly (A) signal (BGH poly (A)) was used. B) HEK-293T cells were transfected with CMV–ΔARE (ΔARE) or CMV-ARE (ARE) plasmids and cultured for 40 hours. Total RNA was extracted with Trizol® reagent. Quantitative one-step RT-PCR assays were then run to measure ARE and ΔARE mRNA. ARE and ΔARE mRNA levels were normalized against GAPDH mRNA levels. Results are expressed as the mean of 4 independent experiments, the error bar represents the standard deviation. C) HEK-293T cells were cotransfected with CMV-ΔARE and pSuper-si20 (ΔARE si20), or with CMV-ΔARE and pSuper-si ctrl (ΔARE si ctrl). Samples were analyzed as in B). D) HeLa Tet-off cells were transfected with pcDNA3 and Tet-ΔARE (ΔARE); pcDNA3; Tet-ARE (ARE); or pSuper si20 and Tet-ΔARE (ΔARE/si20) and cultured for 40 additional hours. Doxycycline was then added to block transcription, and total RNA was isolated with Trizol. Reporter mRNA degradation was analyzed by Northern-Blotting and 18S ribosomal RNA was utilized to control RNA loading. Half-lives were analyzed using the phosphoimager. E) HeLa Tet-off cells were transfected with a total of 5 µg of plasmid DNA. The mass ratio of pSuper-si20 and Tet-ΔARE plasmids were 0, 0.1, 0.2, 0.5, 1, and 2. pcDNA3 was utilized to normalize a total of 5 µg plasmid per transfection. Cells were then cultured for 40 additional hours before total RNA was isolated using Trizol. Reporter mRNA was analyzed by Northern-Blotting and the ratios of 5’frag/Full were analyzed using the phosphoimager.