Insertion of strong poly(A) signal in the intergenic region increases the expression of both c-fms and HMGXB3. (A) SV40-PA is inserted in the intergenic region by CRISPR–Cas9. Confirmation of insertion is shown in Supplemental Figure S3. (B) SV40-PA knock-in abolishes the expression of HMGXB3 mRNA with 3′ end extension, and (C) the expression of c-fms mRNA with 3′ end extension. Northern blot was exposed for 96 h. (D) 32P-labeled strand-specific antisense probe 2 is derived from the c-fms mRNA 3′ end. 32P-labeled strand-specific antisense probe 4 is derived from HMGXB3 mRNA 3′ end. 32P-labeled strand-specific probes 2 and 4 were synthesized as described in Supplemental Figure S1. (E) SV40-PA knock-in increases c-fms mRNA and HMGXB3 mRNA (n = 4). (F) SV40-PA knock-in increases both c-fms protein and HMGXB3 protein. Numbers below bands indicate band intensities scanned by ImageJ. (G) SV40-PA knock-in increases c-fms mRNA half-life, and (H) HMGXB3 mRNA half-life (n = 3).
