Figure 1.
Generation of the PlxnA4KRK-AAA mutant mouse line and the Farp2 KO mouse line. A, qRT-PCR analysis revealed unchanged PlxnA4 mRNA transcript levels in E13.5 PlxnA4KRK-AAA heads compared with WT littermate controls. PlxnA4 KO heads were processed as well as a negative control. Quantification was performed using a relative quantification analysis (ΔΔCt) and presented as mean ± SEM. ***p < 0.001 (Student's t test). B, C, Western blot analysis of E13.5 DRGs and cortices extracted from PlxnA4KRK-AAA and WT littermate controls revealed similar Plexin-A4 and Nrp1 protein levels in both genotypes. Heads of E13.5 PlxnA4 KO were used as a negative control. Data are mean ± SEM. Statistical analysis: Student's t test. D, PlxnA4KRK-AAA, WT littermates, and PlxnA4 KO cortices were subjected to anti-Nrp1 immunoprecipitation and probed using a Plexin-A4 antibody. No change in Nrp1-Plexin-A4 association was detected in the PlxnA4KRK-AAA mutant compared with WT. E, F, 7 DIV cortical neurons from E13.5 PlxnA4KRK-AAA and WT littermate embryos were subjected to a cell-surface biotinylation assay, followed by Western blot probing for Plexin-A4. The total mass of biotinylated proteins in the range of ∼120-260 kDa, detected and quantified using streptavidin-HRP, was used as loading control. Data are mean ± SEM; N.S., Non Significant. G, Primary cortical neurons of PlxnA4KRK-AAA, WT littermates, and PlxnA4 KO embryos were immunostained in culture using a Plexin-A4 antibody. Plexin-A4 protein levels and its spatial pattern in the PlxnA4KRK-AAA mutant were comparable with the WT, whereas virtually no Plexin-A4 was detected in the negative control KO. Scale bar, 25 µm. H, Schematic representation of the Farp2 locus on chromosome 1 (GRCm38-mm10) and the CRISPR-Cas9 KO design. Two sgRNAs flanking an 18,167 bp sequence were used: sgRNA 1 targeted a region upstream of the Farp2 gene promoter, and sgRNA 2 targeted a sequence in intron 2. P1 and P2 (black triangles) represent primers used for detection of the KO allele in a PCR analysis of genomic DNA, targeting sequences outside of the deletion area (upstream of sgRNA1 and downstream of sgRNA2, respectively). Similarly, P3 and P4 (gray triangles) represent primers used for detection of the WT allele and were targeted at intron 1. RT 1 and 2 (blue triangles) represent primers used for reverse transcription analysis of Farp2 gene expression levels; RT 1 targeted a sequence comprised of the 3′ of exon 4 and 5′ of exon 5, and RT 2 targeted exon 6. I, PCR analysis of tail genomic DNA obtained from three E13.5 embryos of a Farp2 Het x Het cross, using primers 1-4 (P1-P4). Lengths of the WT and KO alleles are 551 and 271 bp, respectively. +/+, WT; +/−, Farp2 heterozygous; −/−, Farp2 KO. J, Reverse transcription analysis of WT (+/+) and KO (−/−) cDNA using either Farp2 primers RT 1-2 (top), or Actin β (Actb) primers as internal control (bottom). G, H, Successful KO of the Farp2 gene and its transcripts.