Replacement of Er81 by EWS-Pea3
(A) Generation of Er81EWS-Pea3 mutant mice. Above is the organization of the Er81 genomic locus in the region targeted by homologous recombination in analogy to [14]. Exons 1–4 are shown as light blue boxes, and the Er81 start codon in exon 2 is indicated as ATG. The probe used to detect homologous recombination is shown as a grey box. Below is replacement of Er81 by EWS-Pea3 through the integration of EWS-Pea3 in frame with the endogenous start codon of the Er81 locus in exon 2 (in analogy to [14]).
(B) PCR and Southern blot analysis of Er81EWS-Pea3 wild-type (+/+), heterozygous (+/−), and homozygous (−/−) genomic DNA to detect the mutant allele. PCR primer pairs (EWS-Pea3ki) were used to detect specifically the recombined allele, and a primer pair in exon2 was used to detect the presence of the wild-type allele [14].
(C–E) Analysis of Er81 expression in lumbar DRG neurons of E16.5 wild-type (C), Er81−/− (D), and Er81EWS-Pea3/− (E) embryos. Inset in lower right corner of each panel shows Isl1 expression in the respective DRG.
(F–H) PV expression in lumbar DRG of E16.5 wild-type (F), Er81−/− (G), and Er81EWS-Pea3/− (H) embryos. Confocal scans were performed with equal gain intensity.
(J) Transcriptional transactivation of luciferase expression from a minimal reporter construct containing five consensus ETS DNA-binding sites ( GCCGGAAGC; [18,19]) and a minimal TK promoter upon transient transfection of Er81 (n ≥ 7; 3.03 ± 0.66) or EWS-Pea3 (n ≥ 7; 20.3 ± 2.7). Relative luciferase activity normalized to control (Con).
Scale bar: 80 μm.