Engineered Insulin-Luciferase Fusion Reporter

The insulin luciferase vector was created by insertion of the nanoluciferase cDNA (Promega) in-frame, into the SmaI and BstEII sites of the mouse Ins2 cDNA. We extended the cDNA to the minus 360 base of the genomic DNA sequence to capture the minimal mouse Ins2 promoter. For the mIns2-GFP reporter, we replaced the ATG to stop codon of the preproinsulin-nanoluciferase fusion with GFP using HiFi cloning (New England Biolabs). For the minCMV H2B-GFP shRNA vector, the minCMV GFP polyA signal was cloned from the bidirectional vector minCMV-PGK, a gift from L. Naldini.^{10 (link)} The mini-mir30 based shRNA cassette inserted into the 3' UTR of the GFP was cloned from pSicoR-mCh-mir30, a gift from Michael McManus.^{20 (link)} The U6 sgRNA cassette and dCas9-KRAB cDNA were a gift from Michael McManus.^{21 (link)}