Figure 1.

Deletion of IKKβ in MC3T3–E1 cells. (A) Schematic diagram of the sgRNAs designed to inactivate mouse IKKβ, which were placed inside Ikbkb exon 4. Three PAM motifs were shown in red color. (B) Workflow for generation of mutant cells in MC3T3–E1. Generally, two or more independent sgRNAs were designed and expressed in different vectors which express ECFP or DsRed2 as reporters. 48 h following transfection, cells simultaneously expressing two reporter proteins were subjected to cell sorting by FACS. Genotyping of the mutant clones was done by running electrophoresis with the PCR amplicons on agarose gel. (C) Ikbkb^−/− clone 19, 24, and 26 were selected by PCR and agarose electrophoresis. CRISPR/ Cas9-mediated genomic editing resulted in the PCR products of Ikbkb^−/− clone 19, 24, and 26 around 40 and 150 bp smaller than that of the wildtype (WT) control. H₂O was used as negative control. (D) Representative Western blot for total IKKβ protein levels of MC3T3–E1 WT cells and CRISPR/Cas9-mediated Ikbkb^−/− cells.