Generation of Calm1 long 3′-UTR deletion mice using CRISPR–Cas9. (A) Diagram of the strategies used to eliminate the production of mature long Calm1 3′-UTR transcripts. Six gRNAs (g1–g6) were injected simultaneously to generate a variety of deletions (Deletion 1–3). (B) Calm1 northern blot of adult cortex from three deletion strains and control littermate demonstrating successful deletion of the long Calm1 3′-UTR isoform. Note the Deletion 2 line generates a new isoform with a truncated long 3′-UTR due to the preservation of the distal PAS. Transcripts from the Calm2 and Calm3 genes were found to be unaltered. Psmd4 used as a loading control. (C) Complete loss of Calm1-L in the Calm1ΔL/ΔL (Deletion 3) hippocampal neurons has been confirmed by lacked smFISH signals representing Calm1-L. (D) DIG in situ performed in the Calm1ΔL/ΔL (Deletion 3) embryo also revealed no expression of Calm1-L.
