Generation of mice with a conditional Foxp2 null allele

Abstract

Disruptions of the human FOXP2 gene cause problems with articulation of complex speech sounds, accompanied by impairment in many aspects of language ability. The FOXP2/Foxp2 transcription factor is highly similar in humans and mice, and shows a complex conserved expression pattern, with high levels in neuronal subpopulations of the cortex, striatum, thalamus, and cerebellum. In the present study we generated mice in which loxP sites flank exons 12-14 of Foxp2; these exons encode the DNA-binding motif, a key functional domain. We demonstrate that early global Cre-mediated recombination yields a null allele, as shown by loss of the loxP-flanked exons at the RNA level and an absence of Foxp2 protein. Homozygous null mice display severe motor impairment, cerebellar abnormalities and early postnatal lethality, consistent with other Foxp2 mutants. When crossed to transgenic lines expressing Cre protein in a spatially and/or temporally controlled manner, these conditional mice will provide new insights into the contributions of Foxp2 to distinct neural circuits, and allow dissection of roles during development and in the mature brain.

FIG. 1

Schematic representation of the Foxp2 conditional targeting strategy. Recombination of the targeting vector with the Foxp2 genomic locus results in the introduction of a FLPe-Neo cassette, flanked by FRT sites (ovals), 5′ of exon 12, and leaves two loxP sites (arrows) surrounding the cassette and exons 12–14. The thymidine kinase gene at the 3′ end of the targeting vector enables selection against clones containing randomly integrated vector. FLPe-mediated recombination enables removal of the selection cassette, and generates the floxδneo allele. The δ12–14 allele can be generated from either the floxneo or floxδneo allele by Cre-mediated recombination. PCR genotyping primers and relevant restriction enzyme sites and probes used for Southern analysis are indicated.

FIG. 2

Generation of a Foxp2 conditional allele. (a) Southern analysis of ES cell clone DNA. Left, SpeI-digested genomic DNA was hybridized with a probe to exon 10 (Probe A), mapping beyond the 5′ end of the region included in the targeting vector. * marks an example of a correctly targeted clone, demonstrated by the presence of a 5.6 kb fragment. The WT allele gives a 9.2 kb fragment. Right, SwaI-digested DNA was hybridized with an intronic probe (Probe B) mapping beyond the 3′ end of the region included in the targeting vector. The WT allele gives a 22.6 kb fragment. Appropriate integration of the 3′ loxP site was demonstrated by the presence of an 8.7 kb fragment (clones 1 and 3), whereas recombination within the loxP-flanked region yields a 15.3 kb fragment (clones 2 and 4). (b) PCR genotyping strategy. Two PCR reactions were used to identify the four Foxp2 alleles (cf. Figure 1). The P1/P2/P4 multiplex reaction detects WT, floxneo and floxδneo (top panel), and the P1/P6 primer pair detects δ12–14 (bottom panel).

FIG. 3

Global Cre-mediated deletion of exons 12–14 yields mice that are null for Foxp2. (a) Quantitative real-time RT-PCR using RNA from the striatal precusor region of E16.5 embryos (error bars represent SDs). Data confirm a total loss of Foxp2 exons 13–14 in transcripts from homozygous mutants, and a half-dosage of these exons in heterozygotes. Flanking exons (6–7 and 16) show reduced expression in mutants. Expression of Foxp1, Foxp4, Gad2, and Dlx2 appears normal in mutants. (b) Western blot analysis of lysates from the striatal precusor region of E16.5 embryos using an antibody recognizing the N-terminus of Foxp2. Reprobing with an anti-actin antibody demonstrates equal protein loading. (c) Pup weights (error bars represent SE of the mean). (d) Percentage of pups with both eyes open at P15. (e) Time taken for pups to right themselves after being placed on their backs (error bars represent SE of the mean); note that WT and heterozygote pups are able to right themselves with virtually no delay. (f) Cerebellar morphology at P22 in WT (i, ii), heterozygotes (iii, iv), and homozygotes (v, vi). Homozygotes show reduced cerebellar size and foliation, as shown by whole brains (i, iii, v) and Nisslstained sagittal sections through the vermis (ii, iv, vi). All whole brain photographs were taken at the same magnification.