Key Points
- To determine the functional roles of putative negative control elements and cAMP responsive sequences located in the 5' flanking regions of mouse Ren1d and Ren2d genes.
- Cloned 5' flanking regions of mouse Ren1d and Ren2d upstream of a thymidine kinase promoter fused to a chloramphenicol acetyltransferase (CAT) reporter gene.
- Transfected the chimeric plasmids into mouse pituitary tumor AtT-20 and human choriocarcinoma JEG-3 cells under basal conditions and following stimulation with 8-bromo-cAMP.
- Ren1d 5' flanking sequences in the sense orientation significantly inhibited basal CAT expression, whereas antisense Ren1d and all Ren2d constructs showed no inhibitory effect, indicating the 160-base-pair insertion in Ren2d disrupts negative control function.
- Treatment with 8-bromo-cAMP increased transcription 3-fold for both Ren1d and Ren2d constructs, demonstrating preserved cis-acting cAMP responsive element function in both genes.
Structured PICO
PPopulationMouse pituitary tumor AtT-20 and human choriocarcinoma JEG-3 cells
IInterventionTransfection with chimeric constructs of mouse renin genes (Ren1d and Ren2d) 5' flanking regions fused to CAT reporter gene, and stimulation with 8-bromo-cAMP
CComparatorBasal unstimulated condition; antisense orientation
OOutcomeCAT expression (transcription activity)surrogate
The negative control element is functional in Ren1d but nonfunctional in Ren2d, while the cAMP responsive element is functional in both, providing insight into tissue-specific expression of mouse renin genes.