A partial loss of function allele of
Methyl-CpG-binding protein 2 predicts
a human neurodevelopmental syndrome
Rodney C. Samaco1, John D. Fryer1, Jun Ren3, Sharyl Fyffe1, Hsiao-Tuan Chao2, Yaling Sun1,
John J. Greer3, Huda Y. Zoghbi1,2,4,5and Jeffrey L. Neul4,?
1Department of Molecular and Human Genetics,2Department of Neuroscience, Baylor College of Medicine, One
Baylor Plaza, MS 225, Houston, TX 77030, USA,3Department of Physiology, University of Alberta, Alberta, Canada,
4Section of Neurology, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, MS 225, Houston, TX
77030, USA and5Howard Hughes Medical Institute, Alberta, Canada
Received November 20, 2007; Revised February 20, 2008; Accepted February 27, 2008
Rett Syndrome, an X-linked dominant neurodevelopmental disorder characterized by regression of language
and hand use, is primarily caused by mutations in methyl-CpG-binding protein 2 (MECP2). Loss of function
mutations in MECP2 are also found in other neurodevelopmental disorders such as autism, Angelman-like
syndrome and non-specific mental retardation. Furthermore, duplication of the MECP2 genomic region
results in mental retardation with speech and social problems. The common features of human neurodeve-
lopmental disorders caused by the loss or increase of MeCP2 function suggest that even modest alterations
of MeCP2 protein levels result in neurodevelopmental problems. To determine whether a small reduction in
MeCP2 level has phenotypic consequences, we characterized a conditional mouse allele of Mecp2 that
expresses 50% of the wild-type level of MeCP2. Upon careful behavioral analysis, mice that harbor this
allele display a spectrum of abnormalities such as learning and motor deficits, decreased anxiety, altered
social behavior and nest building, decreased pain recognition and disrupted breathing patterns. These
results indicate that precise control of MeCP2 is critical for normal behavior and predict that human neuro-
developmental disorders will result from a subtle reduction in MeCP2 expression.
Rett Syndrome (RTT, OMIM #312750) is an X-linked
neurodevelopmental disorder characterized by regression of
language and hand use after a period of normal initial cogni-
tive development (1). During this regression, autistic features
can manifest, sometimes leading to the misdiagnosis of
autism. After regression, characteristic clinical features such
as distinctive hand stereotypies, movement abnormalities,
breathing irregularities, autonomic dysfunction, seizures and
sleep disruption become prominent. The disorder primarily
affects girls at a frequency of 1:10 000–20 000 live female
Mutations in Methyl-CpG-Binding Protein 2 (MECP2) are
found in over 95% of typical RTT (3), with ?70% of these
cases caused by point mutations. MeCP2 primarily functions
as a transcriptional repressor (4,5) by recruiting histone dea-
cetylases to DNA that contains the epigenetic mark of
behave similarly to a complete deletion of the coding
sequence of MECP2, indicating that they are complete or
partial loss of function alleles (3). In females, these point
mutations cause features characteristic of RTT; in contrast,
the same point mutations may result in severe infantile ence-
phalopathy and early death in males (7). Additionally, a
number of point mutations in MECP2 not associated with
RTT have been identified in males with moderate mental
retardation, movement abnormalities and psychiatric features
(8–16). These mutations are often found in X-linked mental
?To whom correspondence should be addressed at: One Baylor Plaza, MS 225, Houston TX 77030, USA. Tel: þ1 7137986523; Fax: þ1 7137988727;
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Human Molecular Genetics, 2008, Vol. 17, No. 12
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at HAM-TMC Library on February 21, 2013
retardation (XLMR) families with the female carriers dis-
playing mild mental retardation or learning disabilities.
The discovery that mutations in MECP2 cause the neurode-
velopmental disorder RTT led to the analysis of MECP2 in
other neurodevelopmental disorders with similar clinical fea-
tures. For example, MECP2 mutations have subsequently
been discovered in girls with a number of disorders such as
idiopathic autism (17), Angelman-like syndrome (15) and
mental retardation (18). In addition, MeCP2 expression is
decreased in the brains of individuals with neurodevelopmen-
tal disorders such as autism, Down syndrome, Angelman syn-
drome and Prader–Willi syndrome (19,20). Interestingly,
duplications of the genomic region spanning MECP2 have
been discovered in humans who have neurological abnormal-
ities (21–26). Features of this disorder are typically found in
males and are characterized by hypotonia, cognitive impair-
ment, autistic features and language deficits. Of note, the
human disorder was initially predicted by abnormalities
observed in transgenic mice that over-express MeCP2 2-fold
(27). These results point to the importance of MeCP2
expression levels and suggest that neurodevelopmental dis-
orders may result from either increases or decreases in
To determine whether a slight reduction in MeCP2 level
resulted in phenotypic abnormalities, we made use of a con-
ditional allele of Mecp2 (‘floxed’) that retains a neomycin
selection cassette and polyadenylation sequence within the
30-UTR (28) which disrupts the Mecp2 30-UTR isoform that
predominates in the brain (29). In many instances, retention
of selection cassettes has resulted in the generation of hypo-
morphic alleles of the engineered locus (30). When tested,
we found that the ‘floxed’ allele of Mecp2 caused reduced
expression of both Mecp2 mRNA and MeCP2 protein by
?50% and resulted in a broad spectrum of phenotypic
abnormalities. This demonstrates that precise MeCP2 levels
are critical for neuronal function and that slight reduction
inMeCP2 levels in humans is likely to result in clinical
The Mecp2Floxallele decreases mRNA levels of both Mecp2
isoforms and reduces MeCP2 protein level in the brain
To investigate the molecular consequences of the ‘floxed’
(qRT-PCR) to measure the expression level of Mecp2
mRNA in F1 129S6.B6 Mecp2Flox/yanimals and wild-type
(WT) littermate controls at 26–27 weeks of life. Mecp2 is
expressed as two isoforms that differ in the choice of start
sites (31,32). In the e1 isoform, exon 1 provides the start
site and is spliced to the common exon 3. The e2 isoform is
formed by utilizing the start site in exon 2, which is then
spliced to exon 3. We designed qRT-PCR primers and
probes that either span the exon 1–3 boundary (e1–3) or
span the exon 2–3 boundary (e2–3) to quantify the expression
of these Mecp2 isoforms. Additionally, we designed a
qRT-PCR primer/probe set that is entirely contained within
the common exon 3, allowing us to quantify the total Mecp2
transcript level. We find that the Mecp2Floxallele results in
quantitative real-time PCR
an ?50% decrease in the brain in isoform e1, isoform e2
and exon 3 (Fig. 1A).
To assess whether the reduction in Mecp2 mRNA also
resulted in a comparable decrease in protein expression, we
analyzed whole brain extracts by western immunoblotting
(Fig. 1B) from F1 129S6.B6 Mecp2Flox/yanimals and WT lit-
termate controls at 26–27 weeks of life. We used an antibody
that recognizes the common carboxy-terminus (C-term) of
MeCP2. MeCP2 protein levels are decreased in Mecp2Flox/y
animals compared with WT littermate controls. Quantification
of band intensity normalized to Gapdh revealed that MeCP2
levels in Mecp2Flox/yanimals are decreased by 42% compared
with WT controls (P , 0.001).
MeCP2 is decreased by immunofluorescence but the
cellular pattern is unchanged
As a complementary approach to examine MeCP2 protein
levels and to determine whether the reduction in MeCP2
expression was uniform throughout the whole brain, we
analyzed sagittal sections of brain tissue obtained from
Mecp2Flox/yanimals. Indeed, when identical settings were
used to capture confocal images of a WT littermate control
animal (Fig. 1C) and an F1 129S6.B6 Mecp2Flox/yanimal
(Fig. 1D), the overall MeCP2 expression is markedly attenu-
ated in the mutant animals. Higher power magnification
(inset in Fig. 1C and D) reveals that MeCP2 retains the
same overall punctuate nuclear staining pattern in the mutant
animals as the WT animals, but the expression intensity is
reduced. We observed similar attenuated MeCP2 levels in
animals in the F1 129S6.FVB strain (data not shown).
Mecp2Flox/yanimals have normal survival and slight weight
alterations in certain genetic strain backgrounds
Because the complete absence of Mecp2 results in death
between 8 and 12 weeks of life (28,33), we first assessed
whether the ?50% reduction of MeCP2 in the Mecp2Flox
allele has any effect on lifespan. We did not observe early
mortality in Mecp2Flox/yanimals in either a pure 129S6/
Tac.C57BL/6J (F1 129S6.B6), or an F1 129S6/SvEv-
Tac.FVB/N (F1 129S6.FVB). Furthermore, we did not
observe any of the overt abnormalities seen in the Mecp2–
null animals, such as gross body tremor or hindlimb clasping,
in Mecp2Flox/yanimals. However, on the F1 129S6.B6 back-
ground, the Mecp2Flox/yanimals are ?1 g heavier than WT lit-
termate control animals (Fig. 2A) at 9 and 16 weeks of life.
This mild increase in body weight was not observed in the
F1 129.FVB background (not shown).
Mecp2Flox/yanimals have decreased motor performance
Despite the lack of lethality and absence of overt hindlimb
clasping, we assessed the performance of the mutant mice
on a variety of motor tasks. In the accelerating rotating rod
task, a test for motor coordination and learning, the animal
is subjected to four trials a day for 4 days. The mean latency
to fall for each day is recorded. F1 129S6.B6 Mecp2Flox/y
animals spend less time on the rod on Day 1, indicating a
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baseline deficit in motor coordination (Fig. 2B). Although the
mutant animals continue to fall off the rod sooner than WT lit-
termate controls on all 4 days, they exhibit the expected
increase in the time spent on the rod over the successive
days, demonstrating that they are capable of motor learning.
This problem with motor coordination was not apparent in
F1 129S6.FVB animals (not shown).
The coordination deficit is also observed when the mutant
animals are subjected to two additional motor tasks, the
dowel walking task and the wire hanging task. F1 129S6.B6
Mecp2Flox/yanimals fell off the wire sooner than WT litter-
mate controls (Fig. 2C). The mutant animals also have fewer
number of side touches during the wire hanging task (not
shown) and for the dowel walking task (Fig. 2D). These differ-
ences in motor coordination were not observed in F1
129S6.FVB animals (not shown).
Mecp2Flox/yanimals have decreased pain recognition
but intact pain sensitivity
Individuals with RTT have altered pain thresholds (D. Glaze,
personal communication); therefore, we tested F1 129S6.B6
Mecp2Flox/yanimals to determine whether they have altera-
tions in these neurological systems. In the tail flick assay,
which assesses the pain sensitivity and is based on a nocicep-
tive response (34), a reflex is generated within the spinal
column that does not depend on the pain recognition
systems in the brain. Mecp2Flox/yanimals showed a normal
response to the tail flick assay at 40, 50 and 608C, indicating
that the nociceptive reflex is intact (Fig. 3A). However, they
show an increased latency to respond when exposed to the
hot plate assay,a measurement
(Fig. 3A). This requires the transmission of pain sensation
between the spinal cord and the brain to generate a muscle
response leading to paw withdrawal. Also, 5 out of 15 WT
mice (33%) licked their hindlimbs in response to the heated
plate. In contrast, mutant mice only displayed hindlimb paw
withdrawal. The dissociation between the tail flick and the
hot plate seen in these animals suggests a primary deficit in
pain recognition rather than a problem with the peripheral sen-
sation of pain. When these assays were performed on F1
129S6.FVB animals, similar results were observed with no
difference between mutant WT animals in pain sensitivity,
but decreased pain recognition in the mutant animals (not
of pain recognition
Mecp2Flox/yanimals have decreased acoustic startle
and prepulse inhibition
Sensorimotor gating involves the inhibition of a startle
response to a stimulus when that stimulus is shortly preceded
by a less intense stimulus (the prepulse). Alterations in this
gating system have been characterized in neuropsychiatric
conditions such as schizophrenia (35,36). To assess any
abnormalities in sensorimotor gating in Mecp2Flox/yanimals,
we used the prepulse inhibition assay. In this assay the
animals are exposed to a stimulus (120 dB white noise) and
their startle response is recorded. The animals are also
Figure 1. The expression of Mecp2 is decreased in Mecp2Flox/ymice at both the mRNA and the protein level. The mRNA level as measured by qRT-PCR (A) is
decreased in mutant animals. Notably, the e1 isoform (measured by a probe spanning the exons 1–3 boundary, e1–3), the e2 isoform (measured by a probe
spanning exons 2–3 boundary, e2–3) and the common exon 3 (probe e3) all are decreased by ?50% in Mecp2Flox/ymice compared with wild-type littermates.
The overall protein levels of MeCP2 measured by western blot (B) are decreased in Mecp2Flox/yanimals compared with wild-type littermates. MeCP2 was
detected by an antibody specific to the common carboxy-terminus (a-C-term). The bottom blot in (B) is a loading control probed with an antibody to
Gapdh. Each lane represents biological replicates of the respective genotypes. The decrease in MeCP2 protein level is also observed by immunofluorescence
(C, D). Whereas the expression of MeCP2 is clearly discernable as relatively bright nuclear foci in wild-type animals (C), in Mecp2Flox/yanimals the signal
is weaker (D). The small dashed box shows the cortical region represented at higher magnification in the inset in (C) and (D). The higher magnification
reveals decreased MeCP2 levels, but a similar cellular distribution of MeCP2. The scale bar in the large figure in (C) represents 1 mm and within the inset
represents 10 mm.
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exposed to increasing levels of prepulse (74, 78 and 82 dB)
shortly before the stimulus and their startle response is
recorded. The amount of startle to the stimulus after hearing
the prepulse is then expressed as a percentage of the startle
without the prepulse. F1 129S6.B6 animals have a diminished
startle response compared with WT littermate controls (P ¼
0.001, not shown). However, Mecp2Flox/yanimals show
increased inhibitory gating at 74 and 82 dB (Fig. 3B).
Similar results were observed in the F1 129S6.FVB animals
Mecp2Flox/yanimals have altered social behavior
To assess social behavior, we used the partition test, a test of
social interaction without physical contact (37). In this test, the
test animal is singly housed for 4 days in a standard mouse
cage divided into two equal halves by a partition. The partition
is clear and has multiple holes that allow the mouse to see and
smell the adjacent chamber. On the fifth day of single housing,
an adult conspecific male mouse is placed in the adjacent
chamber. The pair is co-housed for at least 18 h. The once
novel mouse partner is now considered ‘familiar’ by the test
subject. The following day, the time the test mouse spends
at the partition engaged in social interest directed at the
partner mouse is recorded in three sequential 5 min test
encounters: test animal versus the ‘familiar’ mouse, test
animal versus a novel ‘unfamiliar’ mouse and test animal
versus the same ‘familiar’ mouse. The first two test encounters
assess social interest. The last test encounter is a measurement
of social recognition and tests the ability of the test animal to
recognize their original co-house partner. Using this assay, F1
129S6.B6 Mecp2Flox/yanimals spend a larger fraction of the
time at the partition interacting with both the unfamiliar
mouse and the second exposure to the familiar mouse
(Fig. 3C). We have also observed this behavior in Mecp2Flox/y
animals in the F1 129.FVB strain background (not shown).
Mecp2Flox/yanimals have a deficit in nest-building behavior
Previous work demonstrated that another Mecp2 allele
(Mecp2308) shows deficits in nest-building behavior (37);
therefore, we tested this skill in F1 129S6.B6 Mecp2Flox/y
animals. Fourteen hours after being presented with nest build-
ing material, 5 out of 15 mutant animals (33%) showed no nest
building compared with 0 out of 16 of the WT littermate con-
trols. Similarly, only 4 of the 15 mutant animals (27%) had
fully formed nests, compared with 10 of the 16 WT animals
Mecp2Flox/yanimals have hippocampal and
amgydala-dependent learning problems
To characterize the effect of Mecp2Floxallele on learning, we
tested F1 129S6.FVB Mecp2Flox/yanimals and WT littermate
controls on the fear conditioning task. In this task, the animal
is placed in a chamber (the context) exposed to a 30 s sound
pulse (the cue) before receiving a mild electrical shock.
During the training day, the animal is exposed to two cue/
shock pairings. The following day the animal is re-introduced
into the training chamber (context) and the percent time spent
freezing (a fear behavior in mice which indicates memory of
the context) is recorded. The animal is then exposed to a
Figure 2. Mecp2Flox/ymice are heavier and perform poorly on motor tasks. Mecp2Flox/ymice (F1 129S6.B6, n ¼ 16 for each genotype) are ?1 g heavier than
littermate wild-type (WT) controls, although this finding is only significant at 9 and 16 weeks of life (A). The mice perform poorly on a variety of motor tasks
including accelerating rotarod (B), dowel walking (C and D) and wire hanging (C). On the accelerating rotarod, the mutant mice perform poorly on Day 1, which
indicates an inherent coordination deficit. The Mecp2Flox/ymice fall off the wire sooner than controls (C, P ¼ 0.002, Mann–Whitney) but not on the dowel
(P ¼ 0.12, Mann–Whitney). Additionally, the Mecp2Flox/yanimals have fewer side touches in both the dowel task (D, P ¼ 0.013 Mann–Whitney) and the
wire hang task (not shown, P ¼ 0.002 Mann–Whitney).?P , 0.05,??P , 0.01,???P , 0.001.
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chamber with altered visual and odor stimuli. The cue is pre-
sented and the percent time freezing is recorded. Mecp2Flox/y
animals have decreased freezing (Fig. 4A) both when
re-exposed to the context (hippocampal dependent) and to
the cue (amgydala- and hippocampal-dependent). The learning
deficits were not apparent in F1 129S6.B6 animals (not
Mecp2Flox/yanimals have decreased anxiety
F1 129S6.FVB Mecp2Flox/yanimals and WT littermate con-
trols were tested for anxiety and locomotor activity using
the open-field assay. Mutant animals did not show any differ-
ence in the distance traveled, movement time or speed (not
shown) compared with WT animals. However, mutant
animals did have a greater ratio of center/total distance tra-
veled compared with the WT animals in the first and last 10
min intervals (Fig. 4B). The willingness of rodents to
explore the open center of the chamber reflects decreased
anxiety. An additional measure of anxiety is the amount of
vertical exploratory movements that a mouse makes during
the test. Anxious mice perform less vertical explorations
than non-anxious mice. The mutant mice show significantly
more vertical activity during the first and last 10 min intervals
(Fig. 4C) compared with WT mice. Finally, during the task,
mutant mice had more stereotypy counts than WT control
animals (2047 versus 1708 beam break counts, P , 0.05, not
Mecp2Flox/yanimals have altered respiratory patterns
Because girls with RTT (38) and RTT mouse models (39–41)
have disrupted breathing patterns, we analyzed breathing in F1
129S6.FVB Mecp2Flox/yanimals and WT littermate controls at
4 months of life. The respiratory pattern shows qualitative
differences between WT and mutant animals (Fig. 5A and B).
There was a marked increase in the incidence of apneas
(defined as ?1 s duration with at least two missed breaths)
in Mecp2Flox/yanimals (39.5+3.7 per hour) relative to WT
(5.8+0.9 per hour) mice (Fig. 5C). Additionally, the coeffi-
cient of variability of the respiratory rhythm was significantly
higher in Mecp2Flox/yanimals (1.6+0.1) relative to WT
(0.71+0.07) mice (Fig. 5D).
Detailed characterization of the Mecp2Flox/Yanimals revealed
a surprising array of phenotypes associated with subtle altera-
tions of MeCP2 levels. The ‘floxed’ allele of Mecp2 results in
an ?50% reduction of MeCP2 levels, which is likely second-
ary to the presence of the neomycin cassette within the 30-UTR
(28,30). This reduction creates a hypomorphic allele of
Mecp2, which results in a variety of behavioral and physio-
logical changes such as learning problems, movement abnorm-
alities and breathing dysfunction. These results further
underscore the critical importance of the tight regulation of
MeCP2 levels. The phenotypic abnormalities of transgenic
animals with a mild over-expression of MeCP2 (27) and
Figure 3. Mecp2Flox/ymice have altered pain sensitivity, prepulse inhibition, social interactions and nest building. Mecp2Flox/ymice (F1 129S6.B6, n ¼ 16 for
each genotype) have increased latency of paw withdrawal on the hotplate (A, P ¼ 0.002); however, they show normal response to tail flick at 40 (A), 50 and 608C
(not shown). The Mecp2Flox/yanimals have a diminished startle response (not shown, P ¼ 0.001) but increased inhibitory gating when presented with a 74 or
82 dB sound prepulse (B). Mecp2Flox/ymice (F1 129.B6, n ¼ 16 for each genotype) spent a greater percentage of time at the partition than WT littermate controls
when both an unfamiliar mouse was placed in the adjacent chamber or when the mice were re-exposed to a familiar mouse (C,?P , 0.05,??P , 0.01). When
singly housed with a fresh nesting material, many Mecp2Flox/ymice (n ¼ 15) did not show any attempt to form a nest and only a small percentage had a fully
formed nest after 14 h, in contrast to the large percentage (D, x2P ¼ 0.02) of WT littermate control animals (n ¼ 16).
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people with duplications of the MECP2 locus (21–26) indi-
cate that 2-fold increases in MeCP2 levels are detrimental.
The results presented here indicate that reducing MeCP2
levels by ?50% are also detrimental. The critical need for
precise MeCP2 expression levels suggests that this protein
plays a dynamic role in neuronal function rather than a
static role in repression of non-neuronal genes.
Furthermore, the finding that this conditional allele is hypo-
morphic underscores the need to design conditional knock-out
experiments carefully to insure the correct interpretation of the
results. Given that this conditional allele results in a large
array of abnormalities, any analysis of Mecp2 conditional
knock-out animals must also include a commensurate analysis
of the ‘floxed’ allele, preferably using littermate controls.
Without these controls, it will be uncertain if any observed
effect is a direct effect of the conditional knock-out or a non-
specific effect of the hypomorphic ‘floxed’ allele. Equally
important, the non-specific effect of a Cre-transgene should
also be considered and the phenotypic effect of that genetic
engineering event should not be discounted. The most straight-
forward approach to manage such uncertainty is to conduct
these conditional knock out experiments in a manner to
include phenotypic characterization of all possible genotypes.
Of the 11 neurobehavioral assays performed, 8 were per-
formed on two strain backgrounds. Although the hypomorphic
allele caused a variety of behavioral phenotypes on these two
different mouse strain backgrounds, it is noteworthy that some
features were enhanced in one strain and subdued in the other.
These behavioral differences between the strains does not
impact the overall conclusion of the paper that a 50%
reduction of MeCP2 results in behavioral abnormalities,
rather this suggests that such a reduction in MeCP2 is sensitive
to modifier effects. This observation is likely to extend to
humans and suggests that the array of phenotypes associated
with subtle reduction in MeCP2 levels is likely to be broad
and that inter-individual clinical variability will be common.
This work also has important ramifications concerning
potential therapeutic strategies for RTT. Recently, novel com-
pounds that allow translational read-through of premature
stop-codons have been used to treat animal models of muscu-
lar dystrophy (42). Because many common RTT causing
MECP2 mutations create such premature stop codons (3),
interest has developed in utilizing this approach for treatment
of RTT. The difference is that whereas the restoration of 40–
50% of the expression of a structural protein such as dystro-
phin might be sufficient to improve the function of muscles,
functional restoration in RTT might require expression much
closer to WT endogenous levels.
Importantly, this study predicts that human neurodevelop-
mental disorders will result from a decrease of MeCP2
levels by as little as 50%. This decrease in expression may
be the result of sequence changes in the MECP2 locus that
occur either in cis (enhancers, promoter or within the
30-UTR), or possibly via the trans-factors that regulate
MeCP2 expression. This has been suggested by studies that
found reduced MeCP2 levels in the brain of a number of neu-
rodevelopmental disorders (19,20), but the concern has been
that the decreased MeCP2 levels in these post-mortem
samples of neurodevelopmental disorders reflects a non-
specific decrease in neuronal function rather than a specific
finding of the disorders. Other work has identified sequence
polymorphisms in the 30-UTR or MECP2 in individuals with
autism (43–45). The challenge with that work has been estab-
lishing the functional significance of the sequence polymorph-
isms. The work described here demonstrates that a 50%
decrease in MeCP2 levels might indeed cause disease, and
that misregulation of MeCP2 may be a common feature of
many neurodevelopmental disorders.
MATERIALS AND METHODS
Mice were maintained on a 12 h light:12 h dark cycle with
standard mouse chow and water ad libitum. All of the mice
used in these experiments were generated by crossing hetero-
zygous female Mecp2Flox/þmice that had been backcrossed to
129S6/SvEvTac for at least five generations to male mice on
either a pure FVB/N background or male mice on a pure
Figure 4. Mecp2Flox/ymice have altered learning and decreased anxiety. The
Mecp2Flox/ymice (F1 129S6.FVB, n ¼ 16 per genotype) have decreased learn-
ing in a fear conditioning task both when exposed to the context (A, P ¼
0.006) or to the cue stimulus (A, P ¼ 0.004). Additionally, they appear to
be less anxious as measured by the distance traveled within the center of an
open field chamber compared with the total distance (B) during the first and
third 10 min intervals. This decreased anxiety is also apparent by the increased
vertical exploratory movements that the mutant animals under took during the
first and third 10 min intervals in the open field chamber (C).?P , 0.05,
??P , 0.01.
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C57BL/6J background to generate isogenic F1 animals. Only
male animals were used for all the experiments listed with
WT littermate males serving as the controls. Mecp2Flox/Y
and WT littermate controls were co-housed immediately
after weaning. Behavioral experiments were performed with
15–20 mice per genotype with the exception of the open-field
assay, which was performed with 10–12 mice per genotype.
All research and animal care procedures were approved by
the Baylor College of Medicine Institutional Animal Care
and Use Committee.
Accelerating rotating rod. Mice were placed on an accelerat-
ing rotating rod apparatus (Ugo Basile North America, Inc.,
Schwenksville, PA, USA) for 16 trials (four trials per day
for four consecutive days). The rod accelerated linearly from
3.6 to 36 rpm for the first 4.5 min. Each trial lasted a
maximum of 5 min with a 30–60 min inter-trial interval.
The latency for each mouse to fall from the rod was recorded
for each trial. The mean fall time for each day was calculated.
Data were analyzed using a one-way ANOVA (genotype).
Dowel walking. Mice at 11–12 weeks of life were placed in
the center of a 0.9 cm wooden dowel balanced between the
two wooden poles. The latency to fall, the latency to reach
either end of the wooden dowel defined as a ‘side touch’
and the number of side touches were recorded. In the event
of a side touch, the timer was stopped, the side touch recorded
and the mouse was returned to the center of the dowel. Trials
lasted for a maximum of 2 min. Data were analyzed using the
Wire hanging. Mice at 11–12 weeks of life were suspended by
their forepaws on a 2 mm wire and the time remaining on the
wire, the time to first side touch and the number of side
touches, as described above, was recorded. Maximum time
for each trial was 2 min, and similarly to the dowel, whenever
a mouse touched the side wall it was replaced to the center of
the wire. Data were analyzed using the Mann–Whitney U-test
for fall time and number of side touches.
Tail flick. Mice at 16–17 weeks of life were tested for pain
sensitivity using the Tail-Flick Analgesia Meter (Columbus
Instruments, Columbus, OH, USA). Animals were allowed
to acclimate to a plexiglass restraint for 2 min and tails were
placed over the sensing groove. Testing commenced upon
activation of an intense light beam directed at the tails 4 cm
from their base. The latency to observe a tail flick in response
to the light beam was recorded. Each animal was tested using
Figure 5. Respiration is altered in Mecp2Flox/ymice. Representative plethysmographic recordings showing the irregular rhythm and periods of apnea in F1
129S6.FVB Mecp2Flox/ymice (B) relative to WT mice (A). Population data showing the incidence of apneas (C, n ¼ 4 for each genotype). Population data
showing the coefficients of variability of respiratory frequency (D, n ¼ 4 for each genotype).?P , 0.05.
1724 Human Molecular Genetics, 2008, Vol. 17, No. 12
at HAM-TMC Library on February 21, 2013
three different light beam temperature settings (40, 50 and
608C) presented in random order.
Hot plate. Mice at 16–17 weeks of life were tested for pain
recognition using the Hot-Plate Analgesia Meter (Columbus
Instruments). Animals were placed on a 558C heated
surface. A response to the discomfort served as a functional
readout of pain recognition. Responses included hindlimb
licking, shaking or twitching. The latency to respond to the
heated surface was recorded and the data were analyzed
using ANOVA (genotype).
Prepulse inhibition. Mice at 15–16 weeks of life were sub-
jected to acoustic prepulse inhibition. The acoustic prepulse
inhibition task consists of presenting the animal with two
closely paired sound pulses: a prepulse at þ0 dB, þ4 dB
(74 dB), þ8 dB (78 dB), þ12 dB (82 dB) and over back-
ground followed 100 ms later by a pulse of 120 dB. The
amount of startle the pulse induces in the animal is recorded
using a startle chamber for mice (SR-Lab, San Diego Instru-
ments, San Diego, CA, USA) which records activity for
65 ms after the pulse. The maximum amplitude recorded
over the 65 ms is recorded and compared using an ANOVA
(genotype) at each prepulse level.
Partition test. Test mice at 20 weeks of age were individually
housed in standard housing cages for 4 days. Each cage was
separated into two compartments by a perforated barrier
which allows social interaction without direct physical
contact. On Day 5 of individual housing, age- and gender-
matched C57BL/6J partner mice were placed into the com-
partment opposite the test mice. Paired mice were co-housed
in the separate halves of the partitioned cage for at least
18 h. Following this period of induced familiarity, the time
that test mice displayed directed interest in their partner
mice was recorded during three different paradigms: test
subject versus familiar partner, test subject versus unfamiliar
partner and repeated test subject versus familiar partner.
Each behavioral paradigm was assessed during three 5-min
intervals and was performed in sequential order. Data were
analyzed using a one-way ANOVA (genotype).
Nest-building assay. Singly-housed mice at 19 weeks of life
were tested for their ability to build nests. Nest material
(Kimwipes, Kimberly Clark, Dallas, TX, USA) was placed
in each cage 1 h prior to the onset of the dark cycle and
were left undisturbed for 14 h. Nest-building was assessed
based on a three-point scale (1 ¼ 0–25% of material shredded,
2 ¼ less than 50% shredded with material gathered in a nest,
3¼fully shredded) with material gathered in a nest. Data
were analyzed using x2analysis.
Fear conditioning. Mice were tested at 22 weeks of life in a
chamber that contains a grid floor that can deliver an electric
St. Albans, VT, USA). On Day 1 of the test, mice were
placed within the chamber and left undisturbed for 2 min
after which a 30 s white noise sound pulse (‘cue’) was deliv-
ered. At the end of the cue, the mouse was shocked (2 s,
0.4 mA). Two minutes later, a second pairing of sound cue
followed by shock was delivered. Thirty seconds after the
final shock, the animal was removed and replaced in the
home cage. The following day, the animals were replaced to
the same chamber (‘context test’) and freezing behavior was
recorded for 6 min. Freezing behavior was recorded automati-
cally by the instrument. One hour after the context test, the
animals were placed into a chamber which had been cleaned
with an unfamiliar agent (ethanol) and the wall color, the
chamber shape and the odor (artificial vanilla) had been
changed to remove the contextual cues of the chamber. The
animals were then monitored for 3 min. After 3 min, the
white noise cue was started and lasted 3 min. The amount of
freezing was recorded separately for the first 3 min and for
the last 3 min (cue test). The number of freezing intervals
was converted to a percentage of freezing for both the
context test and the cue test, and the data were analyzed
using a one-way ANOVA (genotype).
Open-field analysis. Mice at 12 weeks of life were placed in
the center of chamber (40 ? 40 ? 30 cm) and the activity
was measured by photobeams connected to a computer-
operated Digiscan optical animal activity system (AccuScan,
Columbus, OH, USA). This system measures both XY position
as well as z-activity (rearing). The test was performed with
60 dB white noise and 150 lux illumination. The activity
was measured for 30 min and data were analyzed as three
10 min intervals. The analysis of data was performed using a
one-way ANOVA (genotype).
Quantitative real-time polymerase chain reaction
Freshly dissected whole brains (n ¼ 4 per genotype) from 26–
27-week-old mice were placed in 2 ml Trizol (Invitrogen,
Carlsbad, CA, USA) on ice and immediately homogenized
using a Polytron homogenizer at half maximal. The resultant
homogenates were processed per the manufacturer’s instruc-
tions. RNA was DNAse digested and cleaned using the
Qiagen RNeasy Mini kit per manufacturer’s instructions
(Qiagen Inc., Valencia, CA, USA). First-strand cDNA was
synthesized from 5 mg of the purified RNA using SuperScript
III (Invitrogen). Quantitative PCR was performed using
Applied Biosystems 7300 Real Time PCR System (Applied
Biosystems, Foster City, CA) according to manufactures
instructions using Taqman gene expression assays. Primers
and Taqman probes were designed to assess Mecp2 transcript
levels using the Primer Express v2.0 software program
(Applied Biosystems). The primers and probe sequences are
Mecp2e1–3 (forward primer 50-AGGAGGAGAGACTGG
AGGAAAAG-30, reverse primer 50-CTTTCTTCGCCTTC
TTAAACTTCAG-30; probe 50-FAM-AAGACCAGGATC
Mecp2e2–3 (forward primer 50-GATTCCATGGTAGCTG
GGATGT-30; reverse primer 50- TCTGAGGCCCTGGA
GATCCT-30; probe 50-FAM- AGGGCTCAGGGAGGAA
Mecp2e3 forward primer 50-TACAACCTTCAGCCCA CC
ATT-30; reverse primer 50-CTGAGCTTTCTGATGTTTC
Human Molecular Genetics, 2008, Vol. 17, No. 121725
at HAM-TMC Library on February 21, 2013
TGCTT-30; probe 50-FAM-TGCAGAGCCAGCAGAGGCA
Whole-body plethysmographic measurements of the frequency
and depth of breathing were made from unrestrained male
mice at 12 weeks of life. Pressure changes associated with
breathing were measured with a 260-ml chamber (with fresh
room air flowing at the room temperature of ?238C), a
pressure transducer (model DP103; Validyne Engineering,
Northridge, CA, USA), and a signal conditioner (CD-15; Vali-
dyne). Animals were allowed to acclimate within the record-
ing chamber for 20 min prior to recordings. The mean,
standard error of the mean and coefficient of variability (stan-
dard deviation/mean) were calculated for the respiratory rate
for each subject. Statistical significance was tested using
paired difference Student’s t-test; significance was accepted
at P-values ,0.05.
Freshly dissected whole brains from 26- to 27-week-old mice
were Dounce homogenized in ice-cold RIPA buffer (50 mM
Tris, pH 7.5, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS)
with Roche complete protease inhibitors and 1 mM PMSF.
Samples were rotated for 10 min at 48C and then spun at
maximum speed in a microcentrifuge for 10 min at 48C to
pellet insoluble material. Forty micrograms of each sample
were boiled in a sample buffer, loaded onto a NuPAGE
Bis-Tris 4–12% gel (Invitrogen) and transferred to nitrocellu-
lose for western blotting. Rabbit anti-C-terminal Mecp2
(Upstate, Charlottesville, VA, USA) was used at 1:1000
dilution, and mouse anti-GAPDH clone 6C5 (Advanced
Immunochemical, Long Beach, CA, USA) was used at
1:10 000. HRP-conjugated Anti-rabbit secondary (BioRad,
Hercules, CA, USA) was used at 1:5000 and HRP-conjugated
anti-mouse secondary (GE Healthcare, UK) was used at
Animals were anesthetized (Avertin) and transcardially per-
fused with 4% paraformaldehyde (PFA) for 8 min. Brains
were dissected and post-fixed in 4% PFA overnight at 48C.
After rinsing in 1? phosphate-buffered saline (PBS), brains
were cryoprotected in 30% sucrose and then embedded in
O.C.T. and stored at 2808C. Fifty micrograms of mid-sagittal
sections were cut and suspended in 24-well tissue culture
plates containing 1? PBS. Sections were blocked in 2%
normal goat sera with 0.3% triton X-100 for 1 h at 48C fol-
lowed by a 65 h 48C incubation in a 1:100 dilution of anti-
MeCP2 (Upstate cat# 07–013). Sections were washed four
times for 20 min in 1? PBS and then incubated for 48 h at
48C in 1:500 Alexa 488 labeled goat-anti rabbit (Molecular
Probes). Sections were washed an additional four times for
20 min in 1? PBS and then mounted with ProLong Gold anti-
fade mounting medium (Invitrogen cat# P36930). Images were
collected from optical sections using a Zeiss 510 (Carl Zeiss,
Thornwood, NY, USA) confocal microscope and processed
using ImageJ software (http://rsb.info.nih.gov/ij/).
The authors thank Adrian Bird for the Mecp2Floxmice
S. Maricich for comments on the manuscript and the Baylor
Mouse Neurobehavior Core.
Conflict of Interest statement. None declared.
Autism Speaks (R.C.S.), Cure Autism Now (H.Y.Z.), National
Institutes of Health/National Institute of Neurological Dis-
orders and Stroke (NS052240 to J.L.N.), (NS057819 to
H.Y.Z.), National Institute of Child Health and Human Devel-
opment Mental Retardation and Developmental Disabilities
Research Center (HD024064) and Howard Hughes Medical
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