Figure 4
Deletion in the exon 3 of Igsf3 gene leads near to complete loss of the IGSF3 protein. The CRISPR-Cas9 mediated genome engineering targeted exon 3 of the Igsf3 gene. (A) Schematic presentation of the Igsf3 inactivation strategy. Deleted nucleotides span across the sgRNA binding sites. The red star at the end of the nucleotide sequence indicates the pre-mature stop codon. (B) Gene analysis reveals a 146-nucleotide deletion in the KO allele, which resulted in a premature stop codon right after the signal peptide of Igsf3. PAM protospacer adjacent motif. (C,D) The effect of Igsf3 deletion at the mRNA level was evaluated by using qPCR of the P2.5 cerebrum (C) and intestine (D). In heterozygous tissues, no significant differences in the Igsf3 levels were detected at the mRNA level whereas no Igsf3 transcript was detected in the KO tissue samples. (E) HEK293FT cells were transfected with plasmid encoding the murine Igsf3 gene. Cell extracts were prepared 48 h post-transfection and IGSF3 expression was analyzed using Western blot. WT and mock-transfected HEK293FT were used as controls. Arrow points to the band with correct molecular weight. (F) IGSF3 gene deletion was validated at the protein level from P2.5 cerebrum extracts by using Western blot analysis. The KO showed a nearly complete loss of protein expression while reduced expression was detected in the heterozygous brain samples. (G) The IGSF3 protein expression of the cerebrum samples was quantified by normalizing it to the house keeping protein GAPDH. (H–M) The successful loss of the Igsf3 in the KO animals was further validated by immunostaining the myenteric plexus of the intestinal wall on whole mount samples from P12.5 mice using anti-NCAM1 (H,K) and anti-IGSF3 (I,L) antibodies (N = 3 for each genotype). No IGSF3 signal was detected in the KO intestines (L,M) while expression was clearly visible in the WT (I,J) and it colocalized with the NCAM1. Scale bars: (G–L)100 µm. NCAM levels in (H) and (K) have been enhanced to visualize the neuronal network since NCAM1 expression is reduced in the KO compared to the WT.
