https://openworks.mdanderson.org/sumexp21/1177/thumbnail.jp

Anderson, Matthew

Calin, George A., MD, PhD

Winkle, Melanie, PhD

English

OpenWorks @ MD Anderson

References1. Dragomir, Fuentes-Mattei, Winkle et. al, manuscript in preparation2. Kluiver J, Slezak-Prochazka I, Smigielska-Czepiel K, Halsema N, Kroesen BJ, van den Berg A. Generation of miRNA sponge constructs. Methods. 2012;58(2):113-117.*Figures generated using BioRenderResultsBackground• Sepsis is a severe and life-threatening condition that results from systemic immune dysregulation• miR-93-5p is upregulated in early-stage sepsis and is proposed as a potential targeted therapeuticAims• The luciferase assay can be used to evaluate miRNA binding by comparing florescence levels between miRNA knockouts and controlsHypothesis• We cloned a complementary miR-93-5p sponge construct for use with the luciferase assay in evaluating small molecule inhibitors of miR-93-5p function• We also cloned luciferase constructs containing predicted miR-93-5p binding sites present on the CD28 gene to evaluate miRNA bindingMethodsConclusions• Pilot results suggest that the luciferase assay can be used with Jurkatsuspension cells, however there are low levels of transfection across all cell lines which require optimization• As expected, the Jurkat miR-93-5p knockout expressed lower levels of firefly luciferase activity compared to the wild-type and control lines• Preliminary results suggest that the luciferase assay can be used to evaluate miR-93-5p binding using luciferase constructs, however additional studies are required to confirm and optimize the efficacyFuture Work• Optimization of the transfection process using electroporation, lipofection, or a combination of the two• Following colony PCR validation of the presence of the CD28 insert post ligation, the CD28 constructs will be sent for DNA sequencing and eventual transfection into the Jurkat cell linesAcknowledgementsI would like to thank Dr. Winkle and Dr. Calin for their time and mentorship, as well as the CPRIT-CURE program for hosting this opportunity. I would also like to thank my advisor, Dr. Francis Steiner, for the years of support and continual encouragement to pursue science.1bsmiR-93XhoPmeS/AS5’-TCGAGTAGAGCTCTAGTCTACCTGCACGAACAGCACTTTGGTTT-3’3’- CATCTCGAGATCAGATGGACGTGCTTGTCGTGAAACCAAA-5’A.B.Fig 1. Construct and Subcloning DesignA) Complementary miR-93-5p sponge constructB) Schematic of the subcloning procedure for CD28 binding site inserts into the luciferase vectorA.B.Fig 2. Dual Luciferase Reporter Assay Results Post-Transfection with the miR-93-5p SpongeA) Renilla/Firefly luciferase values across Jurkat cell lines. (n=3, SEM shown)B) Average doubling time of Jurkat cells over several passages (n=6)

Evaluating microRNA Binding Using Luciferase Constructs

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1177&amp;context=sumexp21

Evaluating microRNA Binding Using Luciferase Constructs

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