Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene

Abstract

The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.

Figure 1. p75^NTR targeting vector and screening of ES colonies

a, A schematic presentation of p75^NTR protein domains; the p75^NTR genomic locus depicting a segment from exon 3 to intron 6; the p75^NTR targeting vector and the p75^NTR targeted allele. b, An example of a PCR screen using primers a’ and b’ (red arrows) to identify correctly targeted ES cell clones; positive clones: #15, #18, #53, #87 and #132; ‘+’ and ‘−’ depict the positive and negative controls, respectively ((a’) = 5‘-GGGGATCCGCTGTAAGTCTGCA-3’ and (b’) 5’-TGGGGGAGGGGTGGCTAATTT-3)’. c, PCR analysis using a/b primers (blue arrows in a; (a) 5‘-CCTCCGCCAGCTGTCTGCTTCCT-3’ and a reverse primer (b) 5’-GGGTGGAAGCTGGGACTGTGCACATGC-3’) and template DNA from the putative correctly targeted clones (Fig. 1b) demonstrate that the clones #15 and #53 had retained the 5’ loxP site. The wild type allele produces a 574-bp amplification product. Since the strategy to insert the 5’ loxP site into intron 3 involved the generation of a 207-bp deletion, the mutant allele gives rise to a 407-bp amplification product. d, Verification of the correct targeting using Southern blot analysis. Genomic ES cell DNAs were digested with BamHI and EcoRI and probed with the external probe (red bar in a). Correctly targeted clones #15 and #53 display both the wild-type and targeted alleles of 7.4 kb and 5,9 kb, respectively, while the non-targeted clones #18 and #87 show only the wild-type allele (7.4 kb).

Figure 2. Generation of mice carrying the floxed (p75^NTR-FX) and knockout (p75^NTR-KO) p75 alleles

a, Transgenic EIIa-Cre mice were crossed with mice homozygous for the p75 targeting vector. The resulting mosaic males were further crossed with wild-type females to obtain p75^NTR-FX/WT mice (type I recombination; green arrows c and d depict the primers used [(c) 5‘-TGCAGAAATCATCGACCCTTCCC-3’ and (d) 5‘-TCCTCACCCCGTTCTTTCCCC-3’]; upper panel) and p75^NTR-KO/WT mice (type II recombination; blue and green arrows depict the forward and reverse primers, respectively; upper panel). Tail DNAs were analyzed using PCR to identify samples carrying the floxed allele (lower left panel; primers shown with blue arrows in the upper panel) or the knockout allele (lower right panel; primers shown with blue (forward) and green (reverse) arrows in the upper panel). WT, wild type; HE, heterozygote; HO homozygote. b, RT-PCR analysis of RNAs isolated from brain (B) or liver (L) tissues of 8 week old control and p75 knockout mice demonstrates that p75 knockout tissues did not contain any detectable p75 mRNA (upper panel), while control samples showed the expected 210-bp amplification product. β-actin-specific primers produced a 245-bp amplification product of comparable intensity in all samples (bottom panel). c, Western blot analysis of protein lysates shows that p75^NTR protein is present in wild-type (Wt) brain and liver tissues, while knockout (KO) tissues do not display any detectable protein (arrow points to the p75^NTR protein band).

Figure 3. p75^NTR-KO/KO mice are smaller than their wild-type littermates

a, A p75^NTR-KO/KO mouse is smaller (right) than its control littermate (left) at 4 weeks of age. b, A line graph depicting the size difference between mutant (red line) and control (blue line) mice from 3 weeks of age to 12 weeks of age (3 mice were used in each group).

Figure 4. p75^NTR protein is efficiently ablated from the dorsal root ganglia in p75^NTR/Wnt1-Cre mutants

a, In controls p75^NTR can be detected both in the neural crest-derived dorsal root ganglia (DRG) and in the lateral motor column at E10. a’, higher power view identifying the DRG (circled with the white hatched line). b, In p75^NTR/Wnt1-Cre mutants, p75^NTR is present in the lateral motor column, but not in the DRG. b’ shows the high power image of the region indicated in b. An area circled with the hatched line indicates the DRG region that does not stain positively for p75^NTR protein.

Figure 5. Neurological defects in p75^NTR mutants

When suspended by the tail, both p75^NTR-KO/KO (b) and p75^NTR/Wnt1-Cre (d) mutants display an abnormal hind limb ‘clenching’ response, while corresponding controls (a and c) show a normal ‘stretching’ response. Sciatic nerves of adult (1–3 months old) p75^NTR mutants (f) show about a 30% reduction in diameter when compared to those of control littermates (e). g, Bar chart showing quantitation of the difference in axon diameter between controls and p75^NTR mutants. i, p75^NTR mutant sciatic nerves display a statistically significant lower number of small diameter unmyelinated and lightly-myelinated axons when compared to those of controls (h).