Generation of a novel mouse model
that recapitulates early and adult onset
glycogenosis type IV
H. Orhan Akman1,∗, Tatiana Sheiko1, Stacey K.H. Tay4, Milton J. Finegold2, Salvatore DiMauro5
and William J. Craigen1,3
1Department of Molecular and Human Genetics,2Department of Pathology and3Department of Pediatrics,
Baylor College of Medicine, Houston, TX, USA,4Department of Pediatrics, Yong Loo Lin School of Medicine, National
University Health Systems, Singapore and5Department of Neurology, Columbia University Medical Center, New York,
Received June 4, 2011; Revised and Accepted August 16, 2011
Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the
glycogen branching enzyme (GBE). The diagnostic feature of the disease is the accumulation of a poorly
branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with vari-
able tissue involvement and age of disease onset. Absence of enzyme activity is lethal in utero or in infancy
affecting primarily muscle and liver. However, residual enzyme activity (5–20%) leads to juvenile or adult
onset of a disorder that primarily affects muscle as well as central and peripheral nervous system. Here,
we describe two mouse models of GSD IV that reflect this spectrum of disease. Homologous recombination
was used to insert flippase recognition target recombination sites around exon 7 of the Gbe1 gene and a
phosphoglycerate kinase-Neomycin cassette within intron 7, leading to a reduced synthesis of GBE. Mice
bearing this mutation (Gbe1neo/neo) exhibit a phenotype similar to juvenile onset GSD IV, with wide spread
accumulation of PG. Meanwhile, FLPe-mediated homozygous deletion of exon 7 completely eliminated
GBE activity (Gbe12/2), leading to a phenotype of lethal early onset GSD IV, with significant in utero accumu-
lation of PG. Adult mice with residual GBE exhibit progressive neuromuscular dysfunction and die prema-
turely. Differently from muscle, PG in liver is a degradable source of glucose and readily depleted by
fasting, emphasizing that there are structural and regulatory differences in glycogen metabolism among tis-
sues. Both mouse models recapitulate typical histological and physiological features of two human variants
of branching enzyme deficiency.
Glycogen is a highly branched glucose polymer synthesized by
two enzymes: (i) glycogen synthase (GYS), which attaches
glucose to nascent linear chains of glycogen; and (ii) the glyco-
gen branching enzyme (GBE), which attaches a short branch of
approximately 4 glucose units to the linear chain. Absence of
GBE causes glycogen storage disease type IV (GSD IV,
ically diagnosed histologically by the accumulation of a poorly
branched form of glycogen known as polyglucosan (PG).
In GSD IV, PG mainly accumulates in the liver, heart, skeletal
muscles and the central nervous system, tissues with high meta-
bolic activity. GSD IV is a very heterogeneous disorder, affect-
ing different organs at different ages, with visceral and/or
GBE activity, there are clinically distinguishable forms of the
disease, including an early onset form with no residual
enzyme activity, a juvenile or adult-onset form associated
with partial activity and a clinically distinct adult-onset
disease that is known as adult polyglucosan body disease
∗To whom correspondence should be addressed at: Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor plaza T528,
Houston, TX 77030, USA. Tel: 1 7137988306; Email: firstname.lastname@example.org, email@example.com or firstname.lastname@example.org
# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: email@example.com
Human Molecular Genetics, 2011, Vol. 20, No. 22
Advance Access published on August 19, 2011
disease and is typically due to the absence of GBE activity. The
clinical features include failure to thrive, hepatosplenomegaly
and progressive liver cirrhosis, typically leading to death in
early childhood (1). The neuromuscular presentation of
the disease can be separated into four groups based upon the
age at onset: perinatal, presenting as fetal akinesia and
perinatal death; congenital, with hypotonia and death in early
infancy; childhood, with myopathy and/or cardiomyopathy;
and adult, with isolated myopathy or APBD. APBD was first
sive upper and lower motor neuron dysfunction, marked
distal sensory loss (mainly in the lower extremities), early
neurogenic bladder, cerebellar dysfunction and dementia
(3–7). The typical neuropathologic findings are numerous
large PG bodies in peripheral nerves, cerebral hemispheres,
basal ganglia, cerebellum and spinal cord (5,7,8). Isolated
cases of PG myopathy without peripheral nerve involvement
have also been described.
Currently, no treatment is available for GSD IV, although
liver transplantation has been performed in patients with
apparently isolated liver involvement (9). Two naturally
occurring animal models of GBE deficiency, American
quarter horses and Norwegian forest cats, are not practical
laboratory animals (10,11). Therefore, we developed a mouse
genesis of the disease and to test therapeutic strategies.
Generation of mice lacking GBE
A mouse model of GBE deficiency was generated by introdu-
upstream and downstream of the mouse Gbe1 exon 7 via hom-
ologous recombination (Fig. 1A). A representative PCR ana-
lysis of genotyping is shown in Figure 1B. Gbe1+/Neoanimals
were intercrossed to generate Gbe1neo/neomice, or were bred
to a Flpe-expressing mice strain (GT(ROSA)26Sor-Flpe) in order
to delete the sequences between two FRT sites (represented by
the open triangles in Fig. 1A). PCR analysis using primers that
bind upstream and downstream of exon 7 amplifies a 160 bp
DNA fragment instead of a 3020 bp DNA fragment after dele-
tion of the intervening region containing exon 7 and the phos-
phoglycerate kinase (PGK)-Neomycin cassette. The third
reverse primer located downstream of the 5′FRT site amplifies
wild-type DNA in the same PCR reaction in order to detect het-
erozygous and wild-type litters (Fig. 1C). The resulting hetero-
Figure 1. Gene targeting and molecular characterization of a GBE-deficient mouse model. (A) A partial map of the Gbe1 locus and the targeting vector contain-
ing FRT sites flanking exon 7 before and after homologous recombination in ES cells. Crossing Gbe1+/FRTmice with ROSA 26-flpe mice excises exon 7 from
one allele of the Gbe1 gene by Flpe recombinase. Subsequent breeding of heterozygous mice indicated by Gbe1+/2creates a Gbe1 knockout mouse (Gbe12/2).
(B) Agarose gel electrophoresis shows the genotyping of PGK-neomycin cassette positive and negative mice, primers used for the PCR are indicated in (A) as F
and B. (C) Agarose electrophoresis shows the genotyping of exon 7 knockout, heterozygous and wild-type embryos, the primers used for genotyping are indi-
cated as F, R and B. (D) RT–PCR analysis of the mRNA extracted from neonatal muscle tissue, using primers spanning exons 5–10. (E) The sequence analysis
from exons 6–8 from the RT–PCR product shown in (D) from WT (top) and Gbe12/2(bottom) cDNA.
Human Molecular Genetics, 2011, Vol. 20, No. 224431
total muscle RNA isolated from newborn litters of Gbe1+/2
merase chain reaction (RTPCR) analysis. The PCR primers
were designed to amplify an 810 bp wild-type Gbe1 mRNA
fragment spanning exons 4–10 that are 54 kb apart in
genomic DNA. After FLPe-mediated deletion, a 210 bp long
fragment corresponding to exon 7 was absent from the cDNA
(Fig. 1D and E). RT–PCR products were sequenced to demon-
strate the exact location of the deletion (Fig. 1E).
GBE-deficient embryos are stillborn after a normal
Loss of GBE activity in humans can lead to in utero death
(12–14). Gbe12/2mice likewise die at or soon after birth. In
ordertoanalyze thetimeofdeath ofGbe12/2embryos,weper-
formed timed mating ofGbe1+/2animals. Litter size and geno-
type analysis demonstrate that Gbe12/2pups have normal
embryonic survival. The mice are comparable to heterozygous
and wild-type littermates in terms of size and appearance at
E17.5 and after birth (Fig. 2A and B). Ultrasound analysis of
E20 pregnant females did not identify any abnormalities in
morphology or cardiac function (data not shown). Segregation
of the deletion follows the expected Mendelian distribution:
out of 49 pups, 12 were wild-type, 24 were heterozygous and
13 homozygous. Western blot analysis of muscle extracts
obtained from embryos at E17.5 demonstrated the absence of
GBE protein, while Gbe1+/2and Gbe1+/+animals express de-
tectable GBE protein. In contrast, adult animals harboring the
PGK-Neomycin cassette (Gbe1neo/neo) had a significantly
reduced but still detectable amount of GBE protein in muscle
and liver (Fig. 2D). Reduced GBE protein causes early death
1993–2011) and a Kaplan–Meier curve was plotted (Fig. 2E).
No Gbe1neo/neomice survived beyond 39 weeks.
Effect of the absence GBE on glycogen content
We measured GBE activity in E17.5 embryonic muscle tissue.
There was no detectable GBE activity in the Gbe12/2tissue
(Table 1), while Gbe1neo/neoanimals had a low but detectable
amount of GBE activity (Table 2). Next, we determined the
glycogen content in muscle tissue from the different geno-
types. Gbe12/2mice had a significantly reduced amount of
measurable glycogen. However, in all Gbe1neo/neomice, exam-
ined muscle and liver glycogen content was significantly
greater than in the controls, reflecting a greater accumulation
of the abnormal PG. While the differences in glycogen
content between Gbe12/2and Gbe1neo/neomice may simply
reflect the time required to accumulate PG, it prompted us to
determine the GYS activity and protein levels in the fetal
and adult skeletal muscle samples. GYS activity can be mea-
sured with or without glucose 6-phosphate, which is a strong
allosteric activator of GYS. Surprisingly, higher GYS activity
was observed in Gbe12/2muscle relative to that of controls
when theassay reactionlacked
However, in the presence of 10 mM glucose 6-phosphate, the
GYS activity in Gbe12/2skeletal muscle failed to match
the enhanced GYS activity seen in Gbe1+/+and Gbe1+/2
samples. As indicated in Table 1, the maximal activity of
GYS was significantly lower than in control and heterozygous
littermates, and the difference in responsiveness to glucose 6-
phosphate likely reflects additional as yet unknown regulatory
Figure 2. LackofGBEdoesnotaffectembryonicdevelopment.(A)Embryosat
E17.5. Gbe12/2, Gbe1+/2and Gbe1+/+, indicated by 2/2, +/2 and +/+.
Note no evidence of hydrops fetalis. (B) Stillborn Gbe12/2and Gbe1+/+neo-
malin. (C) Western blot analysis of GBE and GYS1 in muscle extracts obtained
analysis of GBE in adult muscle and liver tissue of Gbe1+/+, Gbe1+/Neoand
Gbe1neo/neo. (E) Kaplan–Meier plot illustrates the incidence of death in
Gbe1neo/neoversus Gbe1+/+mice. P , 0.0001, n ¼ 17.
Table 1. Muscle glycogen content, GBE and GS activity
GS activity (nmol/min/mg)
Without G6PWith G6P
Values represent the Mean+SD,∗P , 0.01 Gbe12/2(n ¼ 5) versus both
Gbe12/+(n ¼ 4) and Gbe1+/+(n ¼ 5).
4432Human Molecular Genetics, 2011, Vol. 20, No. 22
changes to GYS in the absence of GBE activity. Western blot-
ting revealed comparable amounts of GYS1 protein in all three
genotypes (Fig. 2C); Gbe12/2neonates contain detectable
PG. We determined whether glycogen is present in other
fetal tissues. Due to size limitations, we used whole-mount
embryo sections and assessed the glycogen content by histo-
chemistry. Whole embryos were fixed in 10% buffered formal-
dehyde and sections were stained with Periodic Schiff base
(PAS). Before PAS staining, the slides were either untreated
or treated with diastase (alpha amylase). Limited digestion
with diastase degrades almost all structurally normal glyco-
gen, while longer and poorly branched PG remains intact
and thus stains with PAS. Whole cross-sections were exam-
ined by light microscopy and vacuoles in the control neonatal
liver can be seen (Fig. 3A), where glycogen was present. In
contrast, Gbe12/2liver lacked any significant vacuoles,
reflecting an overall reduction in glycogen, but a detectable
accumulation of PG (Fig. 3B). Similarly, following diastase
digestion, glycogen was absent from intercostal muscles of
Gbe+/+newborn pups (Fig. 3C), but PG was readily observed
in Gbe12/2animals (Fig. 3D). In the heart, Gbe2/2newborn
pups exhibited extensive vacuolization (Fig. 3F and H) in
comparison to control mice (Fig. 3E and G), and there was
widespread and prominent PG accumulation (Fig. 3H).
Low GBE activity leads to PG formation in tissues
Clinically, GSD IV is diagnosed by the presence of poorly
branched glycogen that is resistant to diastase digestion.
Therefore, we examined the accumulation of diastase-resistant
PG in multiple organs from 4-month-old Gbe1neo/neomice.
The juvenile and adult form of GSD IV is typically a neuro-
muscular disorder, therefore brain and muscle samples were
examined first. Brain sections of Gbe1neo/neomice had PG
bodies located in the somas and extended processes of
neurons (Fig. 4A and B). Skeletal muscle and heart sections
also showed PG bodies, which were absent in normal
muscle and heart (Fig. 4C–F).
Liver stores glucose in the form of glycogen and uses it to
maintain blood glucose levels between meals, therefore liver
tissue was examined for the accumulation of PG, and
there was a significant amount of PG following diastase diges-
tion compared with control sections (Fig. 4G and H).
However, a similar PG accumulation was not observed in
smooth muscle, kidney or spleen sections (Supplementary Ma-
Accumulated PG is not membrane-bound
Heart and skeletal muscle samples were prepared and exam-
ined by electron microscopy. Heart and skeletal muscle
images show PG bodies of 1 and 5 mm size, respectively.
Magnified images clearly show that PG is not membrane-
bound but free in the cytosol, both in skeletal muscle and
heart (Fig. 5A and B).
Glucose metabolism is subtly altered in Gbe1neo/neomice
A defect in synthesizing normal glycogen and degrading
abnormal PG may be expected to interfere with normal
glucose uptake and release. Therefore, adult Gbe1neo/neo
animals were assessed for their ability to store and mobilize
glucose efficiently by glucose tolerance testing. Figure 6
Table 2. Tissue GBE activity in wild-type and Gbe1neo/neodeficient mice
Values represent the mean+SD, P , 0.001 (n ¼ 6 for each group); ND, not
Figure 3. Gbe12/2newborns accumulate abnormal glycogen in multiple
tissues. Formalin-fixed, paraffin-embedded sections of whole mouse newborns
digested with diastase and PAS stained for glycogen. Control (A) and
Gbe12/2(B) liver sections, note the reduction in cleared areas in the
Gbe12/2liver, reflecting reduction in total glycogen content, while also accu-
mulating PG (×400 magnification). Intercostal muscles control (C) and
Gbe12/2(D) mice (×400 magnification), note the red stained PG. Control
(E) and Gbe12/2(F) heart sections (×40 magnification); boxed area (×400
magnification) from the Gbe12/2tissue exhibits widespread vacuolization
and extensive PG accumulation in comparison to the control (G and H).
Human Molecular Genetics, 2011, Vol. 20, No. 22 4433
shows that Gbe1neo/neoanimals were mildly hypoglycemic
after a 16 h fast compared with controls. After glucose admin-
istration, peak blood glucose values in Gbe1neo/neoanimals
were similar to controls, but glucose values returned to base-
line in ,90 min in Gbe1neo/neomice, while in the control
animals blood glucose decreased more gradually over a 3 h
Catabolism of PG is different in Gbe1neo/neoliver than
in muscle and other tissues
The rapid absorption of glucose and slightly lower blood
glucose levels raised the question of whether the liver in
Gbe1neo/neoanimals can adequately degrade PG that has
been formed during feeding. Animals were fasted for 16 h
and muscle and liver glycogen content assessed by biochem-
ical analysis. The quality of glycogen was tested by PAS stain-
ing after diastase digestion. Glycogen levels were quantified as
percent glucose weight by wet tissue weight (w/w) (Fig. 7A).
Before fasting, Gbe1neo/neoanimals have significantly higher
glycogen content in liver and muscle in comparison to
control animals. After fasting for 16 h, muscle glycogen
content did not change in Gbe1neo/neomice, while control
animals had significantly lower muscle glycogen content,
reflecting the degradation of muscle glycogen. However,
liver glycogen content decreased significantly in Gbe1neo/neo
animals after fasting, indicating that, unlike muscle, Gbe1neo/neo
liver utilized PG during fasting. Histological analysis of
liver confirmed the accumulation and degradation of PG
(Fig. 7B), whereas muscle and heart sections demonstrate
significant amounts of PG even after fasting.
Figure 4. Decreased GBE activity leads to widespread PG accumulation. PG
(red stain) is detected in brain, heart, skeletal muscle and liver sections from
Gbe1neo/neomice. Images are ×400 magnification, except for muscle sections
photographed at ×200 magnification.
Figure 5. PG is not membrane-bound in skeletal muscle and cardiac myofi-
bers. Transmission electron micrograph showing the ultra structure of PG in
skeletal muscle (A) and heart (B). Two micrographs on the right show the
framed areas in higher magnification [10- (top) and 20- (bottom) fold].
Figure 6. Gbe1neo/neomice have lower blood glucose levels before and after a
glucose challenge. Glucose tolerance curves in wild-type (diamond) and GBE-
deficient (square) groups (n ¼ 6 for each group). Error bars for the different
time point values represent the mean+SD, P , 0.01 where indicated (∗).
4434 Human Molecular Genetics, 2011, Vol. 20, No. 22
GBE deficiency is a rare disorder with a very heterogeneous
clinical presentation that appears to be determined in part by
infancy, while mutations that reduce enzyme activity cause
juvenile or adult-onset disease. Juvenile onset disease typically
exhibits liver and/or heart involvement, while adult-onset
nosed as amyotrophic lateral sclerosis, multiple sclerosis or
Alzheimer disease (5,7,15). Here we describe a mouse model
of GSD IV that accurately recapitulates histological this
decreasing the expression of the Gbe1 via transcriptional inter-
ference by the reverse-oriented PGK-Neomycin cassette leads
to a hypomorphic allele with residual enzyme activity and
later onset of disease, yet a pronounced accumulation of PG.
Exon 7 of Gbe1 contains the c-terminus of the alpha amylase
domain and the linker region that connects the alpha amylase
domain to the glycosyl transferase domain. Since exon 7
encodes an in-frame 70-amino acid polypeptide, its deletion
nately, our western blot result does not show the shorter pre-
dicted protein due to a cross-reacting band that obscures the
predicted size. Removal of exon 7 in vivo by FLPe-mediated
recombination leads to Gbe12/2pups that die at birth. In
Figure 7. GBE-deficientlivercandegradePGduringfasting.(A)Liverandmuscleglycogencontentinwild-typeandGbe1neo/neoanimalswasmeasuredbeforeand
after a 16 h fast (n ¼ 5 for each treatment). Glycogen content is expressed as percent glucose w/w in fresh tissue. Error bars represent the mean+SD, P , 0.001
where indicated (∗). (B) Diastase treated and PAS stained liver, skeletal muscle and heart sections from the 16 h fasted wild-type and Gbe1neo/neomice.
Human Molecular Genetics, 2011, Vol. 20, No. 224435
utero development appears unaffected by the mutation. While
total glycogen content is reduced in all tissues, a significant
amount of PG is present. This finding is in contrast to that
reported recently by Lee et al. (13), where a nonsense mutation
The authors reported little accumulation of PG, and attributed
the embryonic lethality that was observed to a developmental
heart defect (ventricular non-compaction) and fetal hydrops,
findings we have not observed. This difference may reflect
strain-specific differences, or, perhaps less likely, a second
linked mutation present in the mutagenized strain studied. The
absence of significant amounts of PG in the strain reported by
Lee et al. is in contrast to the observed accumulation of PG in
postmortem human embryos harboring large homozygous
deletions or point mutations that are predicted to eliminate
GBE activity (16). In these human cases, PG accumulated in
sympathetic nervous system that control postnatal respiration.
heart, as shown in the PAS staining of whole-mount wild-type
E17.5 embryo sections. This demonstrates that there is fetal
used during delivery and after birth in order to provide glucose
until newborns begin feeding. With this in mind, we studied the
steric regulation by glucose-6 phosphate. Allosteric regulation
is believed to supersede the hormonal inhibition mediated by
cellular glucose concentrations. High intracellular glucose
causes glycogen accumulation irrespective of cellular energy
status, as demonstrated in disorders of glycolysis, where,
lar energy deficit, glycogen still accumulates (17,18). We have
found substantial GYS activity in Gbe12/2skeletal muscle
and abundant GYS protein. However, GYS activity in
Gbe12/2mice is considerably less responsive to glucose
6-phosphate-mediated activation in comparison to that of
control mice, suggesting that there is a minimal requirement
for GBE in the allosteric activation of GYS. Lack of glycogen
in tissues in the absence of GYS has been reported previously
(19,20). Unlike GBE, GYS has two isoforms; liver-specific
GYS2 and GYS1 that is expressed in other tissues. GYS2-
deficient mice have near-normal development, whereas 90%
of GYS1-deficient mice die after birth. Thus, given that there
is a single Gbe1 locus, Gbe12/2mice mimic the course of
GYS1-deficient mice, yet, like GYS2-deficient mice, also lack
significant amounts of liver glycogen (but readily detectable
PG). Hence, the early death of GBE-deficient mice is likely
not solely a consequence of an absence of structurally normal
glycogen in the liver. While we did not observe hydropic
embryos or left ventricular non-compaction, the hearts of
inGbe12/2mice will address the molecular basis for this struc-
turally abnormal myocardium.
The PGK-Neomycin cassette used for positive selection in
mouse embryonic stem (ES) cells alters the expression of
Gbe1. Both GBE activity assays and western blotting have
shown that transcription of the Gbe1 gene has been reduced.
This has generated a phenotype similar to the juvenile and
adult forms of GSD IV. Point mutations that decrease
enzyme activity such as Y329S and R545H cause amylopecti-
nosis as a recessive trait. Thus, in order to manifest the
disease, enzyme activity must be ,50% (21). However,
there are rare case reports of manifesting heterozygotes, with
clinical features occurring in the sixth decade of life (22). In
aged humans and animals, PG-like bodies known as corpora
amylacea can be seen in white matter and axons, but the rela-
tionship to GBE activity remains unexplored (23–26).
Gbe1+/2mice will be aged to determine whether a reduction
in GBE can lead to the formation of PG bodies in the brain.
PG is also a component of the Lafora bodies that lead to pro-
gressive myoclonus epilepsy [Lafora disease (LD)]. Lafora
disease is an autosomal recessive disorder that becomes symp-
tomatic in teenage years, with progressive myoclonic epilepsy
leading to death within a decade. LD are found in the pericarya
of neurons and other cell types. The pathogenic mechanism of
Lafora disease does not involve the glycogenolytic enzymes
per se, but is caused by the functional loss of regulatory
enzymes that may indirectly affect glycogenolytic enzymes
(27). Two genes causing LD, EPM2A and NHLRC1 (EPM2B),
have been identified (28,29). An animal model of LD has been
described; however, the exact mechanism of PG formation is
not fully understood (30–32). Thus, breeding GBE-deficient
esis of LD.
Gbe1neo/neomice tolerate fasting and respond to a glucose
bolus in a near normal manner, suggesting that storage of
glucose as glycogen or PG can occur very efficiently, even
with low GBE activity. However, there is a tissue-specific dif-
ference in the ability to degrade PG, as reflected by the persist-
ence of muscle PG after fasting, in contrast to its depletion in
liver. The loss of PG from the liver belies the common belief
that PG is an inert indigestible molecule and suggests that dif-
ferences between hepatic phosphorylase and myophosphory-
lase may account for this observation. We found that PG
can be degraded in liver but not in other tissues. Further
studies will be necessary to compare glycogen breakdown in
muscle of Gbe1Neo/Neomice not only after fasting, but also
after exercise, and after a combination of the two conditions.
These results raise the possibility of treatment strategies that
enhance the physiologic degradation of PG. Therefore, this
mouse model will serve as a useful tool for examining the
biology of PG formation and its degradation, and as a means
for testing possible therapeutic approaches.
MATERIALS AND METHODS
Targeting of Gbe1
The Gbe1 targeting vector was assembled by PCR-amplified
and restriction enzyme-digested fragments from ES 129Sv
mouse DNA. A 1020 bp NheI fragment containing Exon 7
was cloned and modified by inserting FRT oligos upstream
and downstream of exon 7. A PGK promoter-Neomycin resist-
ance cassette flanked by loxP sites was cloned 5′to the FRT
site downstream of seventh exon for positive drug selection.
The final construct contained a 2.1 kb 5′-short recombination
arm composed of a portion of intron 6, the upstream FRT
4436Human Molecular Genetics, 2011, Vol. 20, No. 22
site followed by exon 7, the PGK-Neomycin resistance cas-
sette for positive selection flanked by loxP sites and the FRT
site on the 3′end, followed by a 6.5 kb long recombination
arm spanning intron 7. Finally, a MC1-driven thymidine
kinase-1 gene was incorporated into the plasmid for negative
selection against non-homologous recombination (Fig. 1).
All of the junctions in the final construct were confirmed by
sequencing and restriction enzyme digestion. The NotI linera-
lized vector was electroporated into AB2.2 ES cells by the
mouse transgenic core facility at Baylor College of Medicine
Houston, TX. Selection of ES cells used Ganciclovirwas a
negative selection drug and G418 for positive selection.
Cells surviving in the presence of G418 were screened by
PCR for appropriately targeted integration. Positive ES cell
clones were confirmed by additional PCR and Southern ana-
lyses to contain the two FRT sites to be correctly targeted at
both the 5′- and 3′-ends of exon 7. These ES cells were used
to generate chimeric mice by blastocyst microinjection and
these mice bred to confirm transmission of the targeted allele.
Genotyping wild-type and cre recombined alleles
Tail DNA was isolated by standard techniques and the Gbe1
locus PCR amplified using primers, 5′-AGC TTT GGT TAT
AGA CGA ATC ACT, b, 5′-GTC TAT GTC CAG CAC
AGT ATT AAG GA and c.-5′-TCC TGA AAT GGG ATA
TAT GGG ATA TG. PCR reaction was prepared as described
in the vendor’s protocol (New England BioLabs). Thermal
cycles were programmed for touchdown PCR as follows;
initial 5 min denaturation at 958C followed by touchdown
PCR cycles with the following steps; denaturation at 958C
for 30 min, annealing at 658C decreasing 0.58C every cycle
for the next 20 cycles and 728C extension. After the touch-
down protocol, 20 cycles of regular PCR were carried out
starting with 958C denaturation for 30 s, annealing at 558C
for 30 s and primer extension at 728C for 45 s. Amplified frag-
ments are separated on 2% agarose gel and photographed.
Glucose tolerance testing
Mice from control and GBE-deficient groups were fasted for
16 h. D-Glucose (1.2 mg/g weight) was injected into the peri-
toneum of conscious mice. Blood was obtained from the tail at
0, 10, 40, 100 and 180 min after injection, and the glucose
concentration was determined with an Elite glucose meter
(Glucometer Elite, Bayer).
Preparation of samples for biochemical analyses
Four to 6 months old adult animals and 4-month-old female
mice on the 17th day of pregnancy were anesthetized by Iso-
ThesiaTM(Butler Animal Health Supply, Dublin, OH, USA)
embryos and the tissues of adult animals were immediately
frozen in liquid nitrogen and stored at 2808C for further ana-
lyses. Tissues were cut, weighed and homogenized in the
assay buffers as described below.
For western analysis, a 30 mg of tissue homogenate was sub-
jected to sodium dodecyl sulfate-polyacrylamide gel electro-
membranes and incubated with monoclonal antibodies raised
against human GBE and GYS1 (Origene, Rockville, MD,
USA). Detection was achieved with horseradish peroxidase-
conjugated secondary antibodies and enhanced chemilumines-
Quantification of glycogen
Glycogen content was estimated by measuring glucose
precipitated glycogen from muscle or liver tissue, as described
(33). Samples of frozen muscle and liver tissue (?30–60 mg)
were boiled in 200 ml of 30% (wt/vol) KOH for 30 min with
occasional shaking. After cooling, 67 ml of 0.25 M Na2SO4
and 535 ml of ethanol were added. Next, samples were centri-
fuged at 14 500g for 20 min at 48C to collect glycogen. The
glycogen pellet was suspended in water (100 ml), 200 ml of
ethanol was added and centrifugation as described above
was used to harvest glycogen. This ethanol precipitation step
was repeated, and the glycogen pellet was dried in a Speed-
Vac. Dried glycogen pellets were suspended in 100 ml of amy-
loglucosidase [0.3 mg/ml in 0.2 M sodium acetate (pH 4.8)]
and incubated at 378C for 3 h to digest glycogen. To determine
the glucose concentration in the samples, an aliquot (5 ml) of
digested glycogen was added to 95 ml of a solution containing
0.3 M triethanolamine (pH 7.6), 0.4 mM MgCl2, 0.9 mM NADP,
1 mM ATP and 0.1 mg of glucose-6-phosphate dehydrogenase/
ml. The absorbance at 340 nm was read before and after the
addition of 0.1 mg of hexokinase.
digestion of ethanol-
GBE activity was assayed as described by Tay et al. (14).
Briefly, frozen tissue samples were homogenized in all-glass
homogenizers in nine volumes of 5 mM Tris, 1 mM ethylene
diamine tetra acetic acid (EDTA), 5 mM mercaptoethanol,
pH 7.2 and centrifuged at 9200g for 10 min. Branching
enzyme activity was measured by an indirect assay based on
incorporation of radioactive glucose-1-phosphate (PerkinEl-
mer Life and Analytical Sciences, Boston, MA, USA) into
glycogen by the reverse activity of phosphorylase a (Sigma,
St Lois, MO, USA) as an auxiliary enzyme. At 30, 45 and
60 min time intervals, 5 ml of reaction mix was spotted on
WhatmanwNumber 5 qualitative filter paper (Maidstone,
UK). Unincorporated glucose-1-phosphate was washed for
15 min using three changes of 66% v/v ethanol. Glycogen
bound radioactive glucose-1-phosphate was quantified by
liquid scintillation counter (Packard Instruments, Boston,
GYS activity was measured as described (18). Briefly, 15 mg
muscle extracts homogenized in cold homogenization buffer
(50 mM Tris Acetate, pH 7.8, 20 mM NaF and 2 mM EDTA)
Human Molecular Genetics, 2011, Vol. 20, No. 224437
was incubated 20 and 40 min in assay buffer (4% glycogen,
10 mM UDP-glucose,
Tris–HCl pH 7.8 and with or without 10 mM Glucose
6-phosphate). Ten microliters of the reaction mix were
spotted at each time point onto WhatmanwNumber 5 qualita-
tive filter paper, and the filter papers washed and quantified as
described in the GBE assay protocol.
14C labeled UDP-Glucose, 50 mM
Tissue staining and histochemistry
Tissue sections were prepared from 4-month-old mice, fixed in
10% formalin and embedded in paraffin. Slices of 5 mm were
deparaffinized, and one slide of each sectioned block was
treated with 40 ml of 5 mg/ml a-Amylase (Sigma) for 25 s
in a microwave oven set to 600 watts; slides, washed with
deionized water and oxidized with 0.5% periodic acid for
5 min, stained with Schiff reagent for 15 min and then coun-
terstained in hematoxylin for 15 min and rinsed in tap water.
The slides were then examined by light microscopy (Nikon
Eclipse90i, Melville, NY, USA).
Supplementary Material is available at HMG online.
Our thanks to Doraine Rudman, Pamela Parson and Jim
Barrish at Texas Children’s Hospital and Bilqees Bhatti in
the BCM Comparative Pathology Laboratory for technical
Conflict of Interest statement. None declared.
This work was supported by a Muscular dystrophy Association
Development Grant (MDA10027); the Adult Polyglucosan
Body Research Foundation (APBDRF); and the Intellectual
and Developmental Disabilities Research Center and Digestive
Diseases Center at Baylor College of Medicine. The IDDRC is
funded by award number P30HD024064 from the NICHD. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the Eunice Kennedy
Shriver NICHD or HIH.
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