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.
- SourceAvailable from: J. Michael Bowers[Show abstract] [Hide abstract]
ABSTRACT: Autism is a neurodevelopmental disorder with a multifaceted association between genes and the environment. Currently, in the majority of patients, the etiology of autism is not known and coupled with increasing prevalence rates, along with the high degree of heritability of autism, the development of animal models is crucial for studying and developing therapies for autism. A key characteristic of autism is marked abnormalities in the acquisition and use of language. Thus, to understand and ultimately treat autism is an especially difficult task because no animal produces language, as it is defined in humans. In this review, we will discuss the FOXP family of genes, which are a group of transcription factors that have been linked to both autism, as well as language in humans. Due to the association of language/communication and the Foxp family of transcription factors, animal models with targeted disruptions of Foxp functioning are powerful tools for understanding the developmental signaling pathways that may be vulnerable in autism.Autism-open access. 12/2012; Suppl 1(10).
- [Show abstract] [Hide abstract]
ABSTRACT: DNA double-strand breaks (DSBs) frequently occur in rapidly dividing cells such as proliferating progenitors during central nervous system development. If they cannot be repaired, these lesions will cause cell death. The non-homologous end joining (NHEJ) DNA repair pathway is the only pathway available to repair DSBs in post-mitotic neurons. The non-homologous end joining factor 1 (Nhej1) protein is a key component of the NHEJ pathway. Nhej1 interacts with Xrcc4 and Lig4 to repair DSBs. Loss of function of Xrcc4 or Lig4 is embryonic lethal in the mouse while the loss of Nhej1 is not. Surprisingly, the brains of Nhej1-deficient mice appear to be normal although NHEJ1 deficiency in humans causes severe neurological dysfunction and microcephaly. Here, we studied the consequences of Nhej1 dysfunction for the development of the cerebral cortex using in utero electroporation of inactivating small hairpin RNAs (shRNAs) in the developing rat brain. We found that decreasing Nhej1 expression during neuronal migration phases causes severe neuronal migration defects visualized at embryonic stages by an accumulation of heterotopic neurons in the intermediate zone. Knocked-down cells die by 7 days after birth and the brain regions where RNA interference was achieved are structurally abnormal, suffering from a reduction of the width of the external cortical layers. These results indicate that the Nhej1 protein is necessary for proper rat cortical development. Neurons unable to properly repair DNA DSBs are unable to reach their final destination during the development and undergo apoptosis, leading to an abnormal cortical development.Molecular neurobiology. 10/2014;
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ABSTRACT: Disruptions of the FOXP2 gene cause a rare speech and language disorder, a discovery that has opened up novel avenues for investigating the relevant neural pathways. FOXP2 shows remarkably high conservation of sequence and neural expression in diverse vertebrates, suggesting that studies in other species are useful in elucidating its functions. Here we describe how investigations of mice that carry disruptions of Foxp2 provide insights at multiple levels: molecules, cells, circuits and behaviour. Work thus far has implicated the gene in key processes including neurite outgrowth, synaptic plasticity, sensorimotor integration and motor-skill learning.Current Opinion in Neurobiology 10/2014; · 7.34 Impact Factor
Generation of Mice With a Conditional Foxp2 Null Allele
Catherine A. French,1* Matthias Groszer,1Christopher Preece,1Anne-Marie Coupe,1
Klaus Rajewsky,2and Simon E. Fisher1
1The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
2The CBR Institute for Biomedical Research, Harvard Medical School, Boston, Massachusetts
Received 19 March 2007; Accepted 5 April 2007
Summary: 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 con-
served expression pattern, with high levels in neuronal
subpopulations of the cortex, striatum, thalamus, and cer-
ebellum. 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 recombi-
nation 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 con-
ditional mice will provide new insights into the contribu-
tions of Foxp2 to distinct neural circuits, and allow dissec-
tion of roles during development and in the mature brain.
genesis 45:440–446, 2007. Published 2007 Wiley-Liss, Inc.y
Key words: Foxp2;
speech and language; brain development
There are now multiple examples of disruption of the
human FOXP2 gene, with affected individuals displaying
speech and language deficits of varying severity (Lai
et al., 2001; MacDermot et al., 2005; Shriberg et al.,
2006; Zeesman et al., 2006). The first example to be dis-
covered, and hence the most well studied, is that of the
KE family. A heterozygous missense mutation is inher-
ited by affected members (Lai et al., 2001), who have an
impaired ability to learn and produce the sequences of
coordinated mouth movements necessary for speech; a
condition commonly referred to as developmental verbal
dyspraxia (Vargha-Khadem et al., 2005). These problems
are accompanied by expressive and receptive deficits in
oral and written language (Watkins et al., 2002). Func-
tional neuroimaging studies have shown abnormal pat-
terns of brain activation, including underactivation of
Broca’s area, during language-based tasks (Liegeois et al.,
2003). To date, FOXP2 is the only gene clearly linked to
this aspect of neurological function, providing a unique
involved (Marcus and Fisher, 2003; Vernes et al., 2006).
The FOXP2 protein belongs to a group of transcrip-
tion factors characterized by the presence of a forkhead-
box (FOX) DNA-binding domain. FOX proteins regulate
a diverse variety of processes from early embryogenesis
through to adulthood, and have been implicated in disor-
ders of human or mouse development (Carlsson and
Mahlapuu, 2002). Several domains have been identified
in FOXP2 in addition to the characteristic DNA-binding
motif, including polyglutamine tracts, a zinc finger, a leu-
cine zipper, and an acidic C-terminal region (Wang et al.,
2003). The FOXP2 amino acid sequence is highly similar
across a number of distantly-related vertebrate species;
the human and mouse proteins are distinguished by only
three amino acid substitutions and a single-residue differ-
ence in polyglutamine-tract length (Enard et al., 2002).
Moreover, orthologues of FOXP2 show conserved
expression in equivalent brain structures in humans,
rodents, birds, reptiles, and fish, with notable similarities
in sublocalisation (Fisher and Marcus, 2006; Vargha-Kha-
dem et al., 2005). Key expression sites lie within the cor-
tex (pallium in nonmammals), striatum, thalamus, and
cerebellum. In the mammalian cortex the gene is mainly
expressed in the deepest layers, in the rat striatum it is
enriched in striosomes, while hindbrain expression is
confined to inferior olives, Purkinje cells, and deep cere-
bellar nuclei in all species studied thus far (Bonkowsky
and Chien, 2005; Ferland et al., 2003; Haesler et al.,
2004; Lai et al., 2003; Takahashi et al., 2003; Teramitsu
et al., 2004).
*Correspondence to: Simon E. Fisher, The Wellcome Trust Centre for
Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN,
Contract grant sponsors: Wellcome Trust, NIH.
yThis article is a US Government work and, as such, is in the public
domain in the United States of America.
Published online in
Wiley InterScience (www.interscience.wiley.com).
Published 2007 Wiley-Liss, Inc. genesis 45:440–446 (2007)
These data point to widely conserved functions of
FOXP2 orthologues in distributed vertebrate circuits
involved in sensory processing, sensorimotor integra-
tion, and control of skilled coordinated movements.
Analyses of primate sequence variation suggest that the
precise roles of FOXP2 may have undergone modifica-
tions during human history, perhaps in relation to
speech (Enard et al., 2002). Nevertheless, animal models
will yield crucial insights into the contributions of the
gene to the development and function of relevant neural
circuits, and how they may go awry in cases of disorder.
Gene disruption in the mouse is a highly amenable tool
for addressing this question. Given the complexities of
Foxp2 expression during embryogenesis, postnatal de-
velopment and adulthood, in both neural (Ferland et al.,
2003; Lai et al., 2003) and non-neural tissues (Shu et al.,
2001), a strategy that allows spatiotemporal control of
gene disruption is particularly valuable. Thus, in the
present study, we have generated a mouse line in which
critical exons of Foxp2 are flanked by loxP sites (floxed),
and demonstrated successful Cre-mediated disruption of
It is unfeasible to target the entire Foxp2 locus, since it
spans several hundred kilobases. Instead, we chose to
insert loxP sites around exons 12–14, which encode the
DNA-binding motif (Fig. 1). In addition to removing an
essential functional domain, loss of these exons is pre-
dicted to yield premature termination of protein transla-
tion early in exon 15, because of a frameshift. Of note,
Shu et al. previously targeted a similar region of Foxp2 in
genomic locus results in the introduction of a FLPe-Neo cassette, flanked by FRT sites (ovals), 50of exon 12, and leaves two loxP sites
(arrows) surrounding the cassette and exons 12–14. The thymidine kinase gene at the 30end of the targeting vector enables selection
against clones containing randomly integrated vector. FLPe-mediated recombination enables removal of the selection cassette, and gener-
ates the floxDneo allele. The D12–14 allele can be generated from either the floxneo or floxDneo allele by Cre-mediated recombination. PCR
genotyping primers and relevant restriction enzyme sites and probes used for Southern analysis are indicated.
Schematic representation of the Foxp2 conditional targeting strategy. Recombination of the targeting vector with the Foxp2
MICE WITH A CONDITIONAL FOXP2 NULL ALLELE
genesis DOI 10.1002/dvg
standard knockout experiments, and reported that
replacement of exons 12 and 13 by a neomycin resist-
ance cassette produced a null allele (Shu et al., 2005).
For the present study, Bruce-4 ES cells (Kontgen et al.,
1993), of C57BL/6 origin, were transfected with linear-
ized conditional targeting vector. Clones surviving
selection were screened by Southern blotting for
appropriate integration of the 50and 30loxP sites, and
the neomycin cassette (Fig. 2a). Correctly targeted
clone 1 cells were injected into C57BL/6 albino blasto-
cysts to obtain a male chimera. Breeding to C57BL/6
albino females yielded germline transmission, and
Foxp2floxneo/þheterozygotes were identified by coat
color and PCR genotyping (Fig. 2b).
We removed the neomycin resistance gene, since it is
well established that the presence of this selection cas-
sette can influence expression of the floxed gene and/or
neighboring loci (Lewandoski, 2001). The targeting con-
struct incorporates a FLPe gene, driven by the testes-spe-
cific ACE promoter, which theoretically drives ‘‘self-exci-
sion’’ of the FRT-flanked selection cassette in the testes.
However, we found no evidence that the cassette had
been removed in chimeric offspring, probably because
of insufficient levels of FLPe protein expression. We
therefore mated Foxp2floxneo/þheterozygotes to ACTB-
FLPe hemizygotes [Fig. 1(ii)], which express FLPe ubiq-
uitously under control of human b-actin regulatory
sequences (Rodriguez et al., 2000) and offspring were
genotyped by PCR (Fig 2b). Resulting Foxp2floxDneo/þ
heterozygotes were then bred to C57BL/6 wildtype ani-
mals to remove the ACTB-FLPe transgene from the
floxed strain. Mice carrying the floxDneo allele will be
used for future crosses to transgenic strains expressing
Cre recombinase in a region- and/or temporal-specific
manner [Fig. 1(iii)].
To verify functionality of the loxP sites in vivo, and to
determine if Cre-mediated excision of exons 12–14 pro-
duces a null allele, we crossed Foxp2floxneo/þmales to
Foxp2D12–14/þ;Sox2-Creþ/?females [Fig. 1(iv)]. Sox2-Cre
mice provide an efficient means for deleting loxP-flanked
sequences, particularly when the Cre transgene is car-
ried on the maternal line. In this case excision occurs
throughout the early embryo, in all offspring, irrespec-
tive of whether they receive the transgene (Hayashi
et al., 2003; Vincent and Robertson, 2003). As expected,
these crosses produced general deletion of exons 12–14
in offspring and successfully yielded Foxp2D12–14/þand
Foxp2D12–14/D12–14pups, demonstrated by PCR-based
genotyping (Fig. 2b). Quantitative real-time RT-PCR was
used to analyse Foxp2 expression in the striatal precur-
sor region of E16.5 embryos from such crosses. Three
sets of Foxp2 primers were employed from different
regions of the transcript. Foxp2D12–14/D12–14homozy-
gotes lacked the FOX domain-encoding exons – primer
pair 13/14 yielded no product in these embryos, and a
half-dosage in Foxp2D12–14/þheterozygotes (Fig. 3a). RT-
PCR assays for exons 6–7 and 16 showed reduced
expression levels in Foxp2D12–14/D12–14embryos as com-
pared to wildtype littermates, with intermediate levels
in Foxp2D12–14/þembryos. These data suggest that ei-
ysis of ES cell clone DNA. Left, SpeI-digested genomic DNA was
hybridized with a probe to exon 10 (Probe A), mapping beyond the
50end of the region included in the targeting vector. * marks an
example of a correctly targeted clone, demonstrated by the pres-
ence 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 30end of the region included in the
targeting vector. The WTallele gives a 22.6 kb fragment. Appropriate
integration of the 30loxP 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 iden-
tify the four Foxp2 alleles (cf. Figure 1). The P1/P2/P4 multiplex
reaction detects WT, floxneo and floxDneo (top panel), and the P1/
P6 primer pair detects D12–14 (bottom panel).
Generation of a Foxp2 conditional allele. (a) Southern anal-
from the striatal precusor region of E16.5 embryos (error bars represent SDs). Data confirm a total loss of Foxp2 exons 13–14 in tran-
scripts 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 anti-
body 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 Nissl-
stained sagittal sections through the vermis (ii, iv, vi). All whole brain photographs were taken at the same magnification.
Global Cre-mediated deletion of exons 12–14 yields mice that are null for Foxp2. (a) Quantitative real-time RT-PCR using RNA
FRENCH ET AL.
genesis DOI 10.1002/dvg
MICE WITH A CONDITIONAL FOXP2 NULL ALLELE
ther a significant proportion of transcripts from the con-
ditional allele are unstable/degraded e.g. by nonsense
mediated mRNA decay, or genomic regulatory sequences
between exons 12–14 influence expression of Foxp2. Of
note, mutant mice that carry an early stop codon in
exon 7 of Foxp2 show comparable reductions in Foxp2
mRNA expression levels (Groszer et al., in preparation).
No changes in relative RNA expression were observed
for Foxp1, Foxp4, or other striatally expressed genes,
Gad2 (Katarova et al., 2000) and Dlx2 (Porteus et al.,
1991) at this developmental time (Fig. 3a).
Extracts from the striatal precursor region of E16.5
embroys were analysed by Western blotting using an N-
terminal Foxp2 antibody, to determine the impact of
Cre-mediated deletion at the protein level. A single band
corresponding to full-length Foxp2 was observed in
wildtype extracts and in Foxp2D12–14/þextracts at a
reduced intensity (Fig. 3b). No bands could be detected
in Foxp2D12–14/D12–14extracts; there was no evidence of
any truncated protein resulting from loss of exons 12–14
and termination of translation in exon 15 (Fig. 3b).
Homozygous Foxp2D12–14/D12–14pups have a reduced
body weight when compared with littermates, and typi-
cally die around postnatal day 21 (Fig. 3c). In addition,
they display developmental delays (Fig. 3d) and severe
motor dysfunction, including an impaired righting reflex
(Fig. 3e). Although most of the brain appears grossly nor-
mal, there is a substantial reduction in the size and folia-
tion of the cerebellum (Fig. 3f). By contrast, heterozy-
gous Foxp2D12–14/þpups display no overt abnormal-
ities—they gain weight at the same rate as their wildtype
littermates, do not show significant righting deficits, and
have normal cerebellar size and foliation (Fig. 3c–f). Phe-
notypes of these homozygous and heterozygous animals
are highly consistent with those observed in mutant
mice carrying an early stop codon in exon 7 of Foxp2
(Groszer et al., in preparation). The gross phenotype of
Foxp2D12–14/D12–14homozygotes also recapitulates that
obtained by Shu et al. for their standard targeted knock-
out of exons 12–13 (Shu et al., 2005). However, hetero-
zygotes in the Shu et al. study displayed modest develop-
mental delay and reduced rate of weight gain (Shu et al.,
2005), unlike our Foxp2D12–14/þheterozygotes, which
show no such deficits.
In conclusion, we have generated a conditional
Foxp2 allele and shown that homozygous general dele-
tion of exons 12–14 results in absence of Foxp2 pro-
tein, accompanied by developmental delays, severe
motor dysfunction, and neural abnormalities. Crossing
our floxed line with transgenic mice which express
Cre-recombinase in a spatially and/or temporally con-
trolled manner should circumvent problems associated
with early postnatal lethality and allow investigations
of adult animals. Such studies will facilitate investiga-
tions of Foxp2 function in the various tissues where it
is expressed, including brain, lung, intestine, and cardi-
ovascular system. Most importantly, our conditional
allele represents a powerful tool for dissecting the dif-
ferential contributions of Foxp2 to development and
function of distinct neural networks in the mammalian
central nervous system.
MATERIALS AND METHODS
Gene Targeting and Generation of Mutant Mice
Exon numbering for murine Foxp2 is concordant with
that found for orthologous exons in human FOXP2 (Mac-
Dermot et al., 2005), C57BL/6 DNA containing exons
11–16 of Foxp2 was cloned into the vector pEASY-FLIRT
(Casola, 2004). The resulting construct contained a loxP-
FRT-FLPe-NeoR-FRT cassette between exons 11 and 12,
with a second loxP site inserted between exons 14 and
15. Bruce-4 ES cells were cultured on a mitotically inac-
tive primary MEF feeder layer as described previously
(Torres and Kuhn, 1997). 1.2 3 107ES cells were trans-
fected by electroporation with 25 lg of ClaI-linearized
targeting construct. Colonies surviving G418 (163 lg/
mL active ingredient) and gancyclovir (2 lM) selection
were screened for targeted recombination by Southern
blot analysis. Morula-stage embryos were harvested 2.5
days post coitum from superovulated C57BL/6 albino
females (C57BL/6J-Tyrc-2J, Jackson Laboratories) and cul-
tured overnight in M16 media. The following day,
healthy blastocysts were injected with ES cells and trans-
ferred to pseudo-pregnant CD-1 females. Resulting chi-
meras were bred to C57BL/6 albino mice, to enable
germline transmission to be determined by coat color.
All regulated procedures were carried out under UK
Home Office Project Licence 30/2016.
PCR Genotyping of Embryos and Mice
Genotyping was performed using lysates prepared from
a mouse ear-punch or a small piece of embryo tail. Tis-
sue was digested in 100 lL lysis buffer (50 mM Tris-HCl
pH 8.5, 1 mM EDTA, 0.5% Tween 20, 0.5 lg/mL Protein-
ase K) for 1–2 h at 568C, followed by Proteinase K inacti-
vation at 958C for 5–10 min. Digested samples were
microcentrifuged at full speed for 5 min, and 1 lL of the
resulting supernatant was added to a 24 lL PCR mix con-
taining HotStarTaq polymerase (QIAGEN) prepared
according to the manufacturer’s protocol. The strategy
used to genotype the various Foxp2 alleles is described
in Figures 1 and 2 and used the following primers; P1: 50-
TGTCACGTGTGTAAAAAGTCATCTT-30, P2: 50-GAGCAT
GACAGTGGAATTGAATTAT-30, P4: 50-GTCCACTTGTCC
CTCACTAGTAAAA-30, P6: 50-GGATTAACTATTTCTGGA
ATGCAAA-30. The Cre and FLPe transgenes were geno-
typed using primers; Cre1s: 50-TGATGGACATGTTCAG
Flp1: 50-GTGGATCGATCCTACCCCTTGCG-30, Flp2: 50-
GGTCCAACTGCAGCCCAAGCTTCC-30, yielding fragment
sizes of ?880 and 750 bp respectively. Identical cycling
conditions were used for P1/P2/P4 and P1/P6 PCR
assays; initial denaturation (958C for 10 min), product
amplification (13 cycles at 958C for 30 s, 658C (?0.58C/
cycle) for 30 s, 728C for 45 s, followed by 25 cycles at
FRENCH ET AL.
genesis DOI 10.1002/dvg
958C for 30 s, 588C for 30 s, 728C for 40 s), and final
extension (728C for 7 min). Cre assays used the follow-
ing conditions; initial denaturation (958C for 15 min),
product amplification (35 cycles at 948C for 1 min, 558C
for 1 min, 728C for 1 min), and final extension (728C for
10 min). FLPe assays used the same conditions as Cre
assays except that the annealing temperature was raised
to 708C. DNA fragments were separated by electropho-
resis through 1.5% agarose gels.
Tissue from the striatal precursor region of E16.5
embryos was dissected into RIPA lysis buffer and dis-
rupted by sonication. Lysates were centrifuged at
10,000g for 10 min, and the protein concentrations of
the supernatants were determined by Bradford assay.
Proteins (30 lg per lane) were separated on 12% SDS-
polyacrylamide gels and transferred to PVDF mem-
branes. Membranes were blocked in 5% milk, before
overnight incubation with the primary antibody at 48C.
Goat anti-Foxp2 (N-16) polyclonal antibody (Santa Cruz
Biotechnology), and the loading control, mouse anti-
actin monoclonal antibody (Sigma), were both used at
a 1:2,000 dilution. Rabbit anti-goat (Dako) and goat
anti-mouse (BioRad) HRP-conjugated secondary anti-
bodies were applied at a 1:5,000 dilution for 1 h at
room temperature. Proteins were visualized by chemilumi-
Quantitative Real Time RT-PCR
E16.5 striatal tissue, from three brains of each genotype,
was dissected into RNAlater (Ambion), before being
snap-frozen and stored at ?808C. When required, sam-
ples were thawed and transferred to buffer RLT
(QIAGEN) with b-mercaptoethanol, and then disrupted
using a piston homogenizer. RNA was extracted using an
RNeasy kit (QIAGEN), and included an on-column
DNase digestion step. cDNA was synthesized from 2 lg
RNA using Superscript III reverse transcriptase (Invi-
trogen), according to the manufacturer’s protocol. PCR
amplification was carried out using 25 lL reaction vol-
umes with 12.5 lL of SYBR Green Supermix (BIO-RAD),
0.5 lL of each primer (10 lM) and 1 lL of cDNA. Ther-
mal cycling was performed on the iCycler iQ system
(BIO-RAD) with amplification for 50 cycles at 958C for
15 s, 608C for 30 s, and 728C for 30 s. Melting curve anal-
ysis was performed to exclude amplification of nonspe-
cific products. Relative changes in expression were cal-
culated using the 2?DDCT method (Livak and Schmittgen,
2001), using GAPDH as the internal control and the aver-
age of the wild type samples for each primer pair as the
Mice were deeply anaesthetized and transcardially per-
fused with 4% paraformaldehyde (PFA) in phosphate
buffer. Brains were removed and postfixed for a further
24 h, before being embedded in paraffin. Serial sections
were cut sagittally at 5 lM and stained with cresyl violet.
Whole brains were snap-frozen in liquid nitrogen before
being postfixed for 3 h in 4% PFA. Brains were photo-
graphed with a drop of bromophenol blue for enhanced
The authors thank Khuong Nguyen for generating the
conditional targeting construct, Stefano Casola for pro-
viding the pEASY-FLIRT universal targeting vector, Eliz-
abeth Robertson for advice and supply of the Sox2-
Cre mice, and members of the Robertson/Bikoff group
at the Wellcome Trust Centre for Human Genetics for
technical guidance. We are grateful to Carol Williams
and the staff of the British Heart Foundation Func-
tional Genetics Facility, particularly Dominika Paczoska
and Rathi Puliyadi, for help with mouse colony man-
agement and husbandry. Simon Fisher is a Royal Soci-
ety Research Fellow.
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