Fig 5. Creation of a mouse clock dysfunction cell line model.

A. The core clock gene Bmal1 was mutated (functional knockout) in KPC murine pancreatic cells with CRISPR-Cas9 genome editing technology (BKO) with the frameshift site indicated by the blue line. B. The frameshift mutation was induced upstream of the basic helix loop helix (bHLH) domain (red star). The resulting protein lacked critical downstream elements for functionality, including the PAS-A, PAS-B, and PAC domains. C. Western blot demonstrating the loss of BMAL1 protein across 24 hours for wild-type (WT) and BKO cells (M = marker lane). D. The mean (± standard error) Per1 mRNA expression of synchronized KPC (n = 3 at each time point; black dashed) and KPC-BKO (n = 3 at each time point; grey) cells from ZT0-ZT24. E. Graphs comparing the percent of cells in G1, S, and G2 in WT (n = 3; white) and BKO (n = 3; grey) KPC cells.