Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis
BMC Developmental Biology
Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase-a key enzyme in lacto-neolacto ganglioside synthesis
Chien-Tsun Kuan 0
Jinli Chang 0
Jan-Eric Mansson 1
Jianjun Li 0
Charles Pegram 0
Pam Fredman 1
Roger E McLendon 0
Darell D Bigner 0
0 Department of Pathology and the Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center , Durham, NC 27710 USA
1 Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgren Academy at University of Gothenburg, Sahlgren University Hospital/Molndal , SE-431 80 Molndal , Sweden
Background: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. Results: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. Conclusions: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.
Gangliosides constitute a large group of sialylated
glycosphingolipids (GSLs), which are preferentially (the
concentrations intracellularly are most likely higher)
expressed on the outer leaf of plasma membranes. The
clusters of most negatively charged gangliosides are
associated mainly with membranes of either
hematopoietic progenitors or stromal cells of a variety of
tissues. Functionally, gangliosides influence cell growth
and death, probably because they are involved in the
glyco-mediated assembling of signaling molecules, such
as growth factor receptors or integrins, and cell
adhesion molecules and their ligands [1-4], which further
modulate the signaling pathway [5,6]. Gangliosides help
to determine the microenvironment inside a cell its
physical or chemical properties, local pH, calcium
homeostasis, etc. , which could enhance or abrogate
the biological availability of signaling molecules and
disrupt their interactions. All of these conditions within
the cell influence its ability to regulate cell proliferation
and differentiation and cell-cell contact, as well as
oncogenesis and hematopoiesis. Several lines of research
show that gangliosides serve not only as functional
molecules for cell development and growth, but also as
biological markers for cell sorting or as potential targets
in tumor therapy, because aberrant ganglioside
expression has been known to occur in many cancers, such as
lymphoma, neuroblastoma, glioma, melanoma, breast
cancer, and small cell lung carcinoma [9-13].
Gangliosides are distinguished by the actions of
specific core glycosyltransferases (Figure 1). The specific
roles of gangliosides remain incompletely understood.
Nonetheless, because of the dramatic change in their
expression during neuronal developmental
differentiation and brain morphogenesis, as well as their
prominence in the mature central nervous system (CNS),
gangliosides are assumed to have fundamental roles in
the CNS [8,10].
Specific core glycosyltransferase gene knockout in
mice has proven to be a particularly useful approach for
uncovering the functions of gangliosides in the brain
. In 2003, for example, Yamashita et al. knocked out
GM3 synthase (Siat9 gene, CMP-NeuAc:
lactosylceramide a-2,3-sialyltransferase, EC 22.214.171.124) in the a-series
pathway . Mice that carried the mutation of GM3
synthase remained normal as compared to the wild type.
In the b series, mice with disruption of GD3 synthase
(Sia8a gene, CMP-sialic acid: GM3
a-2,8-sialyltransferase, EC 126.96.36.199) showed a relatively normal phenotype
. When GM2/GD2 synthase (Galgt1 gene,
UDP-Nacetyl-D-galactosamine: GM3/GM2/GD2 synthase, also
Figure 1 Lc3 synthase within the gangliosynthesis pathways. The X in the lacto-neolacto series biosynthesis pathway indicates a block due
to disruption of the Lc3 synthase gene, B3gnt5. Six ganglioside synthesis pathways are shown: globo, gala, neolacto, lacto, a, and b. The
molecules 3-LM1, 3-isoLM1, and 3,6-isoLD1 are shown within the lacto-neolacto pathways. Gangliosides GM1 and GD1a are shown in the
aseries pathway and ganglioside GD1b in the b-series pathway. Ganglioside nomenclature according to Svennerholm .
known as GalNAcT, EC 188.8.131.52) was disrupted , the
mutant mice had no overt abnormalities in appearance
and experienced a nearly normal life span. However,
they did show evidence of dysmyelination and some
axonal degeneration. The reasons for the normal life of
these single gene knockout mice are not clear. It may be
that there is functional overlap such that only a partial
disruption of ganglioside synthesis does not elicit severe
pathology. Thus, double knockout mice have been
generated for further elucidation of ganglioside functions.
In contrast to the relatively subtle phenotypic changes
of mutant mice with a single deletion of the
glycosyltransferase gene, double knockout of the Galgt1 and
Sia8a genes displayed a sudden-death phenotype and a
severe CNS disturbance by lethal, sound-induced
seizures . Likewise, by genetic engineering of both the
Galgt1 and the Siat9 genes, the mutant mice developed
a severe neurodegenerative disease that resulted in
death . The results with DKO mice thus indicated
that both the a-series and the b-series gangliosides play
a pivotal role in stabilizing the CNS. Moreover, when
the GlcCer synthase gene (Ugcg: UDP-glucose:ceramide
glucosyltransferase, EC184.108.40.206) was knocked out, which
disrupted the initial step in most ganglioside
biosynthesis pathways, the mice died at embryonic days 6.5 to
7.5 (days E6.5-E7.5) . Knocking out this gene in the
brain produced severe neural defects and abnormalities
in neural cell differentiation and caused death of all
mice within 24 days, which further underscores the
physiological importance of gangliosides in the CNS [19,20].
We have previously demonstrated that several
structures in the lacto-neolacto-series pathways, such as the
gangliosides 3-isoLM1 and 3,6-isoLD1, were
overexpressed in brain tumors . However, the biological
role of these gangliosides has not been fully elucidated.
The aim of this study was to establish a knockout
mouse model lacking the lactoseries GSLs and to
investigate the biological significance of Lc3 synthase, which
initiates the formation of lacto-neolacto-chain
gangliosides (Figure 1). In our previous attempts to generate
monoclonal antibodies (MAbs) to target the
glioma-specific gangliosides 3-isoLM1 and 3,6-isoLD1, we
immunized mice with these two gangliosides to develop
antibodies against them. However, we isolated only
3-isoLM1- and/or 3,6-isoLD1-specific IgM antibodies,
which are unsuitable for therapy, rather than IgG
antibodies, which are more suitable for therapeutic use. In
the past, it has been difficult to produce high-affinity,
IgG MAbs to glycolipid and ganglioside antigens
because of the prevalence of a primitive, low-affinity
IgM response by most mammals to these usually highly
conserved structures. However, Proias group  has
recently developed transgenic knockout mice in which
the GM2/GD2 synthase enzyme gene (Gal-NAcT) is
knocked out. Such mice have then been used to prepare
high-affinity IgG antibodies against complex gangliosides
in the ganglioside series . Similarly, we knocked out
the Lc3 synthase gene B3gnt5, and immunologically
nave mice lacking lacto-neolacto-series gangliosides
were thus generated for immunization purposes to
produce high-affinity IgG MAbs against the
tumorassociated gangliosides 3-isoLM1 and 3,6-isoLD1.
Lc3 synthase is a member of the
beta-1,3-Nglycosyltransferase superfamily, coded by the B3gnt5
gene on chromosome 16 B1 of the mouse genome.
The mouse cDNA of Lc3 synthase includes an opening
reading frame of 1131 base pairs encoding a protein of
376 amino acids, a type II membrane protein. Lc3 synthase
exhibits strong activity in transferring GlcNAc to the
lactosylceramide for lacto-neolacto-chain ganglioside
biosynthesis (Figure 1), and this activity is regulated during
embryonic development, especially during brain
morphogenesis. In adults, Lc3 synthase is expressed mainly in
spleen, placenta, and cerebellum .
In our study, the null mouse pups, in which the Lc3
synthase gene was disrupted, showed no obvious
phenotypic changes if they survived delivery. However, 11%
died in less than two months, and about 9% of the
surviving offspring developed growth retardation. Over
time, alterations in multiple organs were observed. The
obvious phenotypic changes were not in the CNS, as in
the a- and b-series ganglioside mutant mice, but in
reproductive output and spleen B-cell abnormality.
Here, for the first time, we report those phenotypic
changes from null lacto-neolacto-series knockout mice,
as well as the generation of an anti-IgG response against
the lacto-series gangliosides 3-isoLM1 and 3,6-isoLD1.
Establishment of the knockout model in mice
The lacto-neolacto-series ganglioside pathways should
be completely disrupted by B3gnt5 gene knockout
(Figure 1). We established two knockout models, one by
a conventional method (Figure 2) and one with a
CreloxP system (Figure 3). The models differ in two major
features. For the conventional method, coding region
exon 4 and part of intron 3 were knocked out and
replaced by the neo gene cassette, with the progenitor
background of C57BL/6, whereas only exon 4 was
knocked out by the Cre enzyme for the Cre-loxP model,
with the chimerical background of 129sv and C57BL/6
in the offspring. The targeted embryonic stem (ES) cells
were screened and confirmed by Southern blot analysis
(Figure 4A), and the correct targeting and deletion of
the Lc3 synthase allele was then confirmed by
genotyping PCR and PCR product sequencing (Figure 2, 3).
The complete absence of Lc3 synthase transcripts from
several tissues of null mice was further demonstrated by
Figure 2 B3gnt5 gene knockout, conventional method. Conventional method for targeted disruption of B3gnt5. Given the genomic structure
of the mutant allele, we designed primers spanning exon 4 and inserted a neo gene to distinguish all three genotypes. With exon 4 primers,
knockout mice (-/-) displayed a nonspecific band at about 0.4 kb, as shown in lanes 1 and 2 of the PCR gel, with sequencing confirmation of
nonspecific product. For heterozygous (+/-) and wild-type (+/+) mice, two bands were viewed at 0.4 kb and 0.5 kb. Sequence analysis of the
0.5kb band confirmed that it was exon 4 of B3gnt5. With the inserted neo gene primer pair (N1R, N7R), both homozygous (-/-) and heterozygous
(+/-) mice carried a positive 1.9-kb band, whereas a negative result in this PCR analysis indicated wild type (+/+).
RT-PCR in both adult and fetus (Figure 4B, panels a and
b, respectively), in contrast to tissue results for the Lc3
synthase heterozygote and wild type. In the wild-type
adult, Lc3 synthase was strongly expressed in spleen, while
weakly expressed in liver and brain (Figure 4B.a).
Heterozygotes expressed less Lc3 synthase in spleen, which is
consistent with one copy of the gene being knocked out.
In an E17 fetus, both head and body showed strong
expression of Lc3 synthase in wild-type (Wt/Wt) and
heterozygous (Wt/KO) mice (+/+ and +/-, respectively, in
Figure 4B.b). However, tissue from the Lc3 synthase
homozygote, whether it was an adult or a fetus, showed
no transcripts at all (Figure 4B).
To explore whether there was any residual activity of
Lc3 synthase in knockout mice, we quantitated the
lacto-neolacto ganglioside series (Table 1). 3-LM1 and
3-isoLM1 are the major downstream components in the
lacto-neolacto-series pathways (Figure 1). The total
ganglioside content (expressed as mol sialic acid per
gram of tissue) and the distribution of the major
gangliotetraose gangliosides GM1, GD1a, GD1b, and GT1b,
which constitute about 90% of the total ganglioside sialic
acid content, were used as internal positive controls to
exclude any major effect on ganglioside topography.
There were no significant differences among the three
genotypes, KO/KO, Wt/KO, and Wt/Wt, either in the
total ganglioside concentration or in the distribution of
the major ganglioteraose-series gangliosides (GM1,
GD1a, GD1b, and GT1b), in cerebellum or cortex tissue
(Table 1). This indicated that the ganglioside
biosynthesis in the a- and b-series was not affected by the
B3gnt5 gene knockout. The results from adults showed
that the cerebellum expressed a detectable amount of
the neolacto-series ganglioside 3-LM1, about 0.24
nmol/g of tissue for wild-type mice, and Lc3 synthase
heterozygous knockout mice expressed about half that
amount, 0.14 nmol/g of tissue. In contrast, the Lc3
synthase homozygous knockout mice were totally lacking
in 3-LM1 expression. The results from cortex revealed
that only the Lc3 synthase wild-type mice expressed
3-LM1, about 0.18 nmol/g of tissue. Notably, the
wildtype fetus showed 10-35-fold higher expression of 3-LM1
than did the adult, even in comparing results for whole
brain in the fetal assay against results for cerebellum in
Figure 3 B3gnt5 gene knockout, Cre-loxP method. Cre-loxP method for targeted disruption of B3gnt5. For the Cre-loxP model, two primer
pairs were used also. The exon 4 primer pair was the same as that used for conventional knockout (Figure 2). Results were analyzed as for the
conventional knockout method. Another primer set detected the inserted loxP site. Both homozygous (-/-) and heterozygous (+/-) mice carried a
positive 2.1-kb band, which resulted from exon 4 deletion by Cre recombinase. A 4.3-kb band indicated when the targeted portion was not
deleted by Cre recombinase. No band appeared in this PCR analysis for the wild-type mice (+/+). The PCR products were sequenced to further
confirm the homogeneity.
the adult assay. This is consistent with RT-PCR results,
indicating that the wild-type fetus contains more Lc3
synthase. Conversely, we detected no 3-isoLM1 in either
wild-type adult or fetus (Table 1), possibly because
3-isoLM1 is a very minor component of the total series of
gangliosides and, with its relatively low concentration,
might not be detectable by our method.
Phenotypic changes of Lc3 synthase knockout mice
Early stage growth delay
Once the knockout models were established, we
maintained colonies through cross-breeding of heterozygotes.
Of the pups that survived 5 days, about 80% showed
normal postnatal development, in terms of suckling, righting,
and eye opening. However, about 9% of the pups showed
growth delay, or dwarfism, especially in the first 20
postnatal days. Genotyping revealed that, in the total of 12
mice with the dwarf phenotype, only 2 were homozygous
and 2 wild type, and 8 were heterozygous. Additionally, we
completed a genotype analysis of all five pups in one
particular litter, which included one dwarf that was
heterozygous, and four littermates that displayed normal
growth rate and represented all three genotypes, indicating
that the dwarf phenotype may not be directly related to
knockout of the Lc3 synthase gene B3gnt5. This group of
4 littermates served as the same-litter control that is
represented in Figure 5. We noted that the delayed-growth
group could be further divided into two patterns: One
pattern showed growth delay in the early stage after birth,
within about 20 days, and then the growth in these pups
gradually caught up to that of the normal mice (Figure 5,
dwarf group A); the other pattern showed growth arrest at
about 10 days, and these pups then died some time after
that (dwarf group B).
Lower survival rate after birth
The genotype distribution studies on all offspring
identified by PCR showed that, for fetuses from both the
conventional and the Cre-loxP models, heterozygous
Figure 4 B3gnt5 gene knockout analysis. A. Genomic Southern blot of ES cell clones. Genomic DNA from wild-type ES clones and possible
candidate ES clones carrying homologously recombinant B3gnt5 allele were digested with the Xba I restriction enzyme and hybridized to the
genomic probe. Two bands are shown, a 5.8-kb band of wild type and a 2.9-kb band (from Clones C10 and A5), indicating that the
recombination occurred. B. Disruption of Lc3 synthase gene expression shown by RT-PCR. Disruption of Lc3 synthase gene expression shown by
RT-PCR for the wild type (+/+), heterozygote (+/-), and homozygote (-/-). Total RNA was extracted from different organs in both adult and fetus
and reversely transcripted into cDNA. PCR was performed by using cDNA as a template and primers located in exon 4 of Lc3 synthase. Beta
actin was the internal quality control (right panels). a. Results from three different tissues in adult mice. In the wild-type (+/+) genotype, Lc3
synthase gene expression was detected mainly in spleen and was weakly positive in brain and liver. Lc3 synthase expression was decreased in
the heterozygote (+/-) and completely knocked out in the homozygote (-/-). b. Results from head and body of E17 fetuses. Lc3 synthase gene
expression was detected in both head and body tissue in the wild-type (+/+) fetus and the heterozygote (+/-) but was not detected in either
head or body of the homozygote (-/-).
Table 1 Ganglioside concentration of Lc3 synthase knockout mice*
Tissue and Genome Type
KO, knockout; ND, not detected; Wt, wild type; %SA, percentage of sialic acid.
*All values are means SD. The lacto-series ganglioside assay was based on lipid extracts corresponding to 5 mg of tissue that were assayed by
TLCimmunostaining. Detection limits were as follows: 3-LM1, 0.04 nmol/g tissue; 3-isoLM1, 0.04 nmol/g tissue.
Four samples were in each group for the cerebellum assay and the cortex assay. Two samples were in each group from different parental pairs for the fetal
#Remaining gangliosides constituting 100% of total sialic acid: GM3, GM2, GD3, GD2, and GQ1.
Data from one mouse detected.
Figure 5 Early-stage growth delay of dwarfism in mice following Lc3 synthase knockout. The breeding was set in a
heterozygoteheterozygote (+/-, +/-) base. Pups were weighed periodically. Mice of normal weight were weaned at day 21, and mice weighing <10 g were
kept in the parental cage. The average weight of the same-litter control differs significantly from that of the dwarf B group (P = 0.0008), but
differs less from that of the dwarf A group (P = 0.1072). Composition of the three mouse groups: Same-litter control (): one +/+, two -/+, and
one -/-; dwarf A group (): two -/+; dwarf B group (): two -/+.
breeding produced offspring at the expected Mendelian
frequency, 14%-25% for wild type, 29%-32% for
homozygote, and 43%-57% for heterozygote (Table 2). However,
in mature offspring, the survival rate for homozygous
mice showed a 50% drop11%-16% (mature offspring)
versus 29%-32% (fetus), whereas the survival rate for
wild-type mice increased from 14%-25% (fetus) to
32%-50% (mature offspring). One notable feature for the
Cre-loxP model was that in the total 251 surviving pups,
50% were of the wild type, which was twofold higher
than the expected 25% Mendelian frequency (P <
0.0001). This phenomenon became more pronounced in
mice that died at the age of 2 months. About 71% of
these dead mice were homozygotes with no wild type,
and they displayed no symptoms, which indicates that
Lc3 synthase homozygous knockout mice were more
predisposed to death in early life (KO/KO genome in
Table 2). We suspect that the deaths were due to
earlystage developmental defects.
In the heterozygous-heterozygous breeding, we found
that the survival rate of pups after birth was significantly
lower than that from normal mouse breeding. The
survival of offspring from conventional knockout mice
dramatically decreased, to a rate of about 59%, versus the
expected 100% survival rate from normal mouse
breeding, with a P value of < 0.0001 (Table 3A). Most of
those pups died during delivery or immediately after
birth. Although the Cre-loxP model showed a slightly
higher survival rateabout 89%, statistical analysis still
revealed a significant difference versus the expected
Table 2 Genome typing distribution in three groups of
Lc3 synthase knockout mice*
100% survival rate from normal mouse breeding, also
with a P value of < 0.0001.
In further study, we confirmed the significant decrease
in survival rate, both in the conventional model and in
the Cre-loxP model (Table 3). Also, given that the
survival rate of the Cre-loxP model was slightly higher (Table
4A), this further study showed the survival rate dropped
from 89% to 56%, which was similar to the conventional
knockout model, only 16 pups delivered by 13 breeding
pairs in 5 months. We hence inferred that the reduction
in survival rate of the offspring resulted from knockout
of the Lc3 synthase gene B3gnt5.
To compensate for the reduced survival rates, we
increased the breeding pairs in order to obtain enough
pups for further investigation. As a result, in 6 months,
18 breeding pairs of Lc3 synthase heterozygous
knockout mice produced a total of 42 litters, with an average
of 2.3 litters per breeding pair, and an average of 3
surviving pups in each litter (Table 4A). These results
demonstrated a small litter size and lower pregnancy
rate than the average theoretical numbers for mouse
breeding. Further, in these 18 breeding pairs, 6 pairs
lost fertility after the first litter, which indicated a short
reproductive span. Moreover, further breeding showed
deeper defects in both Lc3 synthase knockout models,
with average of 1 litter per breeding pair in 5 months
(1.4 for conventional knockout [19 breeding pairs]; 1.3
for the Cre-loxP knockout method [13 breeding pairs]).
Also, the average surviving litter size further decreased
to 1.6 pups per litter (26 litters) for the conventional
model and 0.9 pups per litter (10 litters) for the
CreloxP system (Table 3). In addition, about 30% of the
dams died during delivery.
Because all of the breeding described in Tables 3 and
4A was carried out with the heterozygote base, we next
examined whether homozygotes could mate and
produce progeny. According to Mendels law, homozygote
breeding could produce only homozygote offspring. Our
genotype results (Table 4B) were consistent with this
conclusion. However, the surviving litter size continued
to decrease (an average of 0.3-1.7 pups per pregnancy, n
= 16 for the two models combined). For the
conventional knockout group, nearly no pups survived any
delivery (average = 0.3, n = 9). Although the Cre-loxP
model produced a few more live pups per pregnancy
(average = 1.7, n = 7), we still concluded that, compared
to the Lc3 synthase wild-type mice, the heterozygotes
and homozygotes (Table 4A and 4B) from both Lc3
synthase knockout models showed severe reproductive
We noted that most offspring died immediately after
birth or during delivery because of the death of dams,
in either heterozygote or homozygote breeding.
Table 3 Survival rate and reproductive effects of Lc3 synthase knockout: Heterozygote breeding analysis in the
*The breeding was carried out with a heterozygote-heterozygote base. The cages were observed periodically, and the parameters shown in column 1 were
recorded. Only the bodies seen dead were counted as dead mice in litter size determination.
About 30% of the females died during delivery for both models.
The percentage was compared to the theoretical number 100%. P < 0.0001 by binomial test.
To examine the possibility of fetus death in utero, we
also performed embryonic studies. The exact date of
pregnancy was judged by positive plug formation. At
days E15 to E18, the pregnant females were dissected,
and the embryos were counted. We found that the total
fetus number per pregnancy was within normal range,
with an average 6.8 to 7 fetuses per pregnant female
(Table 4C). Although we did find 1 to 3 dead fetus
bodies in each of the 5 dams in a total of 29 breeding
pairs, we assumed that the death of the fetuses was not
caused by embryonic lethality. Thus, our embryo
experiments shown in Table 4C led us to conclude that Lc3
synthase knockout mice were fertile and that the major
reasons for reproductive defects had to do with
Table 4 Survival rate and reproductive effects of Lc3 synthase knockout
A. Heterozygote breeding analysis in Lc3 synthase knockout mice*
Outcome of Breeding Conventional Knockout
Number of delivered offspring 230
Number of surviving mice (survival rate) 135 (59%)
Number of breeding pairs 18
Breeding time (months) 6
Number of litters 42
Average litter #/breeding pair 2.3
Average # of pups/litter (av # surviving/litter) 4.7 (3)
B. Homozygote breeding analysis of Lc3 synthase knockout mice
Outcome of Breeding Conventional Knockout
*The breeding was carried out with a heterozygote-heterozygote base. The cages were observed periodically, and the parameters shown in column 1 were
recorded. Only the bodies seen dead were counted as dead mice in litter size determination.
The percentage was compared to the theoretical number 100%. P < 0.0001 by binomial test.
**Pregnancy was indicated by a positive plug and a big belly, but the litter size is sometimes zero here.
At days E15 to E18, fetuses were obtained through dissection of the dam as described in Materials and Methods.
difficulties in delivery or reduced neonatal viability, the
same reasons that ultimately contributed to the overall
reduced neonatal survival rate. We could not find a
good explanation for the short reproductive span and
low pregnancy rate.
We observed alopecia, or fur loss, in both Lc3 synthase
knockout models that affected all regions of the mouses
body (Figure 6A and 6B). The genome typing of these
alopecic mice indicated that the lesions occurred in all
three genotypes, and the associated P values indicated
that the differences in results for the three groups were
not significant (Figure 6A). Because of the fur loss,
some mice acquired skin inflammation, which may have
been caused by scabbing. In addition, the fibrous tissues
that formed during the healing of these wounds resulted
in contractures that prevented mice from fully extending
their legs and necks. We sent mice with inflammation to
a veterinary diagnostic laboratory at Duke University to
test the possible causes. All pathogens for which the
mice were tested, including bacteria and viruses, were
negative. Generally speaking, the alopecia did not affect
the life span of mice, and some of them grew fur again,
albeit with less density. However, some of these mice, if
the inflammation affected movement, died earlier than
Late-stage obesity in male knockout mice
To our surprise, in some male homozygous and
heterozygous mutant mice, we observed an age-related obese
phenotype at about 5 months, these obese mice weighing
almost twice as much as their littermates (Figure 6C).
We did not observe the same phenomenon in female
mutant mice, even at the age of 2 years. About 26% of
the heterozygous males and 40% of the homozygous
males developed the obese phenotype (Figure 6A) (P <
0.05 and P = 0.01, respectively), which indicated that the
Lc3 synthase gene B3gnt5 probably was one of factors
that controlled for lack of obesity. Since not all of the
null mice developed obesity, we presumed that either Lc3
synthase was not the only factor causing the obese
phenotype or the deletion of the Lc3 synthase gene made
mice more sensitive to developing obesity. In general,
obese mice survived a normal life span of about 2 years.
We noted that an increasing number of mutant mice
died in 5-15 months, in contrast to the life span of the
wild-type littermates. Gross morphological examination
revealed splenomegaly and enlarged lymph nodes as
major pathological changes.
B-cell abnormalities of Lc3 synthase knockout mice
Our results from RT-PCR analysis and the ganglioside
assay (Figure 4B and Table 1), in agreement with the
literature , demonstrated that Lc3 synthase was
expressed mainly in the developing embryo of wild-type
mice, and it could not be detected in embryos at the
same stage from homozygous knockout mice.
Postnatally, the expression of Lc3 synthase was restricted to
wild-type spleen and brain, with particularly high
expression in spleen (Figure 4B). As expected, Lc3
synthase expression was completely knocked out in
B3gnt5 homozygous knockout mice (Figure 4B and
Table 1). We compared the mouse brain histology from
the three genotypes of Lc3 synthase, KO/KO, Wt/KO,
and Wt/Wt, in an extensive side-by-side evaluation of
hematoxylin & eosin (H&E)-stained, frozen-section,
whole-mount slides of brain, which were cut in the
horizontal plane to include cerebellum, hippocampus, basal
ganglia, cerebrum, and optic neuron. This analysis failed
to reveal any abnormalities of neuronal migration or
white matter volume (results not shown). We also did
an immunohistochemical analysis using anti-calbindin
antibody, and the anti-calbindin results demonstrated
no significant differences in the three genotypesthe KO
homozygote, the Wt/KO heterozygote, or the Wt
homozygotefor immunoreactivity in cerebellum, cortex, or
spinal cord (results not shown).
The preferential expression of Lc3 synthase in spleen
prompted us to study its specific role in this organ. The
high expression of Lc3 synthase in wild-type spleen and
the evidence of splenomegaly and enlarged lymph nodes
in B3gnt5KO/KO mice prompted us to further study its
specific role in cellular regulation. The overall structure
of wild-type spleen, with H&E staining, showed normal
distribution of splenic nodules (white pulp), germinal
center, and red pulp. However, in the heterozygous and
homozygous B3gnt5 knockout spleens, the marginal
zone disappeared, and the limits between the
disorganized germinal center and the red pulp were blurred.
We also observed a greater amount of white pulp in
homozygotes, with some fused to each other, which
resulted in enlarged white pulp. In addition, for some
splenic nodules, a germinal center architecture
disturbance or a missing germinal center was found in
B3gnt5KO/KOmice (Figure 7).
Because of the histological changes in the spleen of
knockout mice, we examined cell classification in
knockout spleens and in wild-type spleens by
determining the B-cell and T-cell populations in the three
different genotypes. For this study, we used flow
cytometric analysis with cell-type-specific antibodies. As
shown in Figure 7, the B-cell number detected by the
anti-CD19 antibody was reduced significantly, by 39%
(67.6 vs. 41.5, P < 0.001), in homozygous and in
heterozygous B3gnt5 knockout spleens, when compared to
the B-cell number of the wild-type spleen. In contrast,
we found that the population of T cells detected by
anti-CD4 or anti-CD8 antibodies and the population of
NK cells detected by the anti-CD49b antibody were
Figure 6 Alopecia and obesity in Lc3 synthase knockout mice. A. Incidence of alopecia and obesity. A weight of >40 g in a male was
counted as obesity. B. Alopecia. Fur loss was seen in all three genome types (wild-type data not shown) and could occur anywhere on the
mouses body. The arrowhead indicates where the fur loss caused difficulty in extension of hind limbs, and the arrows show fur growing again
with less density. C. Late-stage obesity. Mice were weighed periodically. In panel b, the 24.6-g control with a genotype +/- was from the same
litter as the obese mouse.
Figure 7 Spleen morphology of Lc3 synthase-knockout mice. Mouse spleens from three different genome types of similar age were
sectioned and stained. The wild-type controls included a littermate (panel a) and a mouse from a non-knockout colony (panel b). White pulp is
indicated by a white arrow in panel a, and the green arrow indicates red pulp. A germinal center is indicated by a white arrow in panel b. In
both Wt/KO and KO/KO genotypes (panels c and d) tissue, the arrowhead indicates fused white pulp. The germinal center in the KO/KO mouse
spleen (panel d) is not clearly defined.
Immunization of B3gnt5 Knockout Mice with 3-isoLM1
Our original purpose in developing Lc3 synthase
knockout mice was to create an immunologically nave host
for ganglioside 3-isoLM1 and/or 3,6-isoLD1
immunization, so that high-affinity IgG antibodies could be
produced. Two hypotheses may be stated as possible
explanations for the ability of these mice to produce
high-affinity IgG to gangliosides: (1) The absence of
synthesized endogenous complex gangliosides makes the
mice immunologically nave, or (2) the lack of complex
Mouse Spleen Immunophenotype Analysis*
KO, knockout; Wt, wild type.
*All values are means SD of 5 mice for wild type, 2 for Wt/KO, and 2 for KO/KO.
The P value was obtained through a comparison by Students t test of the wild-type group to either the Wt/KO or the KO/KO
Figure 8 Spleen immunophenotyping of Lc3 synthase-knockout mice. Mouse spleen immunophenotype analysis showing the fluorescence
intensity of the staining of four antibodies: The intensity from T-cell antibodies (CD4 and CD8) was normal in the Wt/KO or KO/KO phenotype
as compared to that in the Wt/Wt phenotype; that for NK cell antibody (CD49b) in the Wt/KO or KO/KO phenotype was similar to that of the
Wt/Wt phenotype, with P 0.05; and that for B cells (CD19) in the Wt/KO or KO/KO phenotype was reduced by about 39% when compared to
that of the Wt/Wt phenotype, with P < 0.001.
gangliosides affects immune regulation such that these
knockout mice produce IgG instead of IgM antibodies.
The B3gnt5 homozygous knockout mice were immunized
with purified gangliosides, either 3-isoLM1 or
3,6isoLD1, conjugated to Salmonella minnesota as a carrier
and adjuvant. Mice were minimally immunized s.c. 5
times with the ganglioside conjugates, and sera from the
immunized mice were tested by ELISA for reactivity
against the purified gangliosides. We have observed
positive antibody responses of the IgG type in the immunized
mice, and pre-immunized mouse serum was used as the
control baseline (data not shown). For both types of
ganglioside immunization, midpoint titers from some of
the sera (post 3 and post 5 immunizations) are near
the 1:10,000 titer range, which is appropriate for
progressing to hybridoma generation (Figure 9). It should also be
noted that these are IgG subclass titers. In virtually all past
attempts, including a variety of different immunization
schemes and numerous strains of immune competent
mice (e.g., C57BL/6, CH3, and SJL/j), to generate
ganglioside-reactive hybridomas, only IgM antibodies were
generated. Nevertheless, the use of the immunologically nave
knockout mice has enabled us to successfully induce IgG
responses to 3-isoLM1 and 3,6-isoLD1 in this
immunization strategy. However, in the course of evaluating them
by making a hybridoma fusion of spleen cells with
myeloma cells, we found that although the fused hybridoma
cells could survive in hypoxanthine aminopterin thymidine
(HAT) medium, very few positive clones were identified,
and these proliferated with low efficiency (data not
The mouse Lc3 synthase gene B3gnt5 was first cloned by
Henion et al. in 2001, whose work indicated that the key
function of the Lc3 synthase enzyme is the regulation of
lacto-neolacto-series ganglioside synthesis during
embryonic development and brain morphogenesis .
Postnatally, the expression was restricted to splenic B
cells, the placenta, and cerebellar Purkinje cells, where it
co-localized with HNK-1 reactivity . We sought to
further elucidate the role of this enzyme.
In the present study, we created two genetic models
by disrupting the B3gnt5 gene, and thus the expression
of Lc3 synthase. We used two different strategies to
establish these knockout mice and then compared the
phenotypic changes in these two models. Both models
showed that lack of Lc3 synthase resulted in complete
deficits in downstream gangliosides of the lacto-neolacto
cascade. Nevertheless, mice with totally deficient
lactoneolacto-series gangliosides experienced normal
embryonic development and were born at the expected
Mendelian ratio, and if they survived delivery, could be
kept viable postnatally. However, about 9% of all
offspring displayed a dwarf phenotype, and 11% died in
Figure 9 Anti-IgG response against 3-isoLM1 and 3,6-isoLD1. Four B3gnt5knockout mice, each shown as a separate curve, were
immunized with purified gangliosides 3-isoLM1 and 3,6-isoLD1 that had been conjugated to Salmonella minnesota as a carrier and adjuvant.
Sera from the immunized mice were tested by ELISA for reactivity against the purified gangliosides (3-isoLM1 and 3,6-isoLD1) as optical density
readings at 492 nm (OD 492 nm).
early life, and genome typing results confirmed that the
Lc3 synthase-null mice tended to die early.
Mice carrying the homozygous deletion of Lc3
synthase were viable. Breeding of homozygous mutant
mice produced live pups, albeit with reduced survival
rate, which may indicate reproductive defects. Recently,
Biellmann et al. reported the disruption of the B3gnt5
gene in mice, and breeding of heterozygous mice failed
to produce any viable homozygous progeny . They
believe that the B3gnt5 gene is essential to
pre-implantation development of the murine embryo. The reason for
the discrepancy with our results is not clear. We
characterized our knockout mice by Southern blot analysis,
RT-PCR, and PCR, and by sequencing genomic DNA
clones, confirming that exon 4 of Lc3 synthase of
homozygous mice was deleted. Most recently, Kondo et al.
from Furukawas group also reported B3gnt5-deficent
mice (B3gnt5-/-) were viable, showing by sensitivity
analysis that GSLs are not pivotal receptors for Subtilase
cytotoxin in vivo with such mutant mice lacking lacto/
neo-lacto series GSLs .
As our mutant mice matured, pleiotropic phenotypic
changes occurred, including dwarfism (Figure 5), fur
loss (Figure 6B), and obesity (Figure 6C). A vexing issue
for either dwarfism or fur loss was the lack of a
satisfactory explanation for these phenotype changes, which
occurred not only in mutant mice, but also in the
wildtype littermates. The fur loss could not be ascribed to
fighting between mice, and this phenotypic change has
not been reported in similar studies of ganglioside
knockout mice. However, a dwarf phenotype in
knockout mice has been reported in several studies
[19,26,27], such as those with knockout of the gene
encoding acetylcholinesterase and Ugcg, encoding
GlcCer synthase, which catalyzes UDP-activated glucose
to ceramide. Those genes knocked out were totally
different from the B3gnt5 gene we knocked out here. The
systemic disruption of Ugcg in mice is lethal during
early embryogenesis, and the offspring with this specific
gene deficiency in brain gained less weight than their
controls in the first 16 postnatal days. Because the
deaths of all homozygous mice that occurred within 24
days in those studies were associated with abnormalities
in neural cell differentiation, it is difficult to draw any
conclusions with regard to the dwarf phenotype from
Of interest in our study was that in the wild-type mice
that were not littermates of a knockout group we could
not identify any similar phenotypic changes. These data
led us to conclude that the dwarfism in wild-type
littermates, differing from the non-knockout control colony,
may have been caused by some genetic background
differences, occurring in germ line transmission, or may
have resulted from knockout techniques or
recombination processes. Recently, Reed and colleagues compiled
body-weight information on about 2000 viable knockout
mice, and they reported about 31% of the knockout
mice weighed less than controls. They assumed that
more than 6000 genes could contribute to mouse body
weight . Thus, it is worth emphasizing that Lc3
synthase may be involved in some way during
development or in the maintenance of body weight.
As for defects in reproductive capability, considerable
data has been accumulated to date about the biological
effects of gangliosides on fertility. Male mice in which
cerebroside sulfotransferase (CST, EC 220.127.116.11) has been
knocked out lose fertility because of a block in
spermatogenesis, while females are able to breed . Similar
observations have been made for mice in which
ceramide galactosyltransferase (CGT, EC 18.104.22.168)  and
GM2 synthase (Galgt1)  are knocked out. Our data
definitely indicate that the absence of
lacto-neolactoseries gangliosides affected the reproductive system, but
predominantly in the female mice. Although the lack of
Lc3 synthase did not appear to affect the capability for
fertilization, after their first pregnancy, at least 33% of
the females lost fertility permanently, even though
breeding was continuously performed. This would
suggest that a short reproductive span results from Lc3
synthase knockout. In our further study, we observed
that the average birth rate was only about one litter per
breeding pair. Thus we could not exclude the possibility
that Lc3 synthase knockout progressively disrupted
fertility, despite normal fertilization in the first pregnancy.
We also investigated the effect of lack of Lc3 synthase
on the pups development in utero. We expected that
the Lc3 synthase gene would be essential for embryonic
development, as indicated by the literature .
However, this was not the case. Random heterozygous
cross-breeding gave an expected Mendelian inheritance,
identified by genome typing (Table 2, Fetus group). The
general morphology of newborn pups was normal (data
not shown). Thus, Lc3 synthase knockout was not lethal
in fetal development. However, delivery was seldom
successful, in that about 30% of the dams died during
delivery. Dissections after the death of the dam usually
revealed 5-7 fully developed, but dead fetuses in the
uterus. Moreover, even if the dams overcame the
difficulties of delivery, about 60% of the pups died
immediately after birth, and about 71% of the dead pups were
KO homozygotes (Table 2). The morphology of dead
newborn pups displayed no significant abnormalities
that might indicate the cause of death.
Our analysis of the phenotypic changes described
above, considered in the context of the relevant
literature, subsequently led us to investigate the role of
lactoneolacto gangliosides in spleen. Although the existence
of clustered gangliosides in the plasma membrane in
human lymphocytes has been shown by electron
microscopy , the functions of these gangliosides have not
been delineated completely. Some evidence indicates
that they have several regulatory effects on monocytes
and myeloid cells , as well as T cells of the immune
system . It is well known that spleen is a secondary
immune organ. We observed enlarged spleen and lymph
nodes in dead mice and identified the spleen to be the
organ with the major Lc3 synthase expression in adult
wild-type mice (Figure 4B). Thus we hypothesized that
these changes in the spleen indicated a relationship to
the biological roles of gangliosides.
Remarkably, we found that depletion of Lc3 synthase,
conceivably abolishing the formation of lacto-neolacto
gangliosides, led to a decrease of B-cell numbers in
spleen in Lc3 synthase-null mice. Also, the spleen
morphology in null mice was changed, with some germinal
centersthe major B-cell locationtotally gone. At first,
because we did not fully understand the significance of
these morphological changes, we continued to employ
the Lc3 synthase-null mouse as a host for the
lactoneolacto ganglioside immunization according to our
experimental design. Using the knockout mouse model,
we elicited an IgG response, but with moderate or low
titers, and the hybridoma fusion of immunized spleen
cells using the conventional polyethylene glycol fusion
method had low viability. During the fusion
experiments, we observed that the fused cells survived in
HAT medium for several months, but did not proceed
to proliferation. Thus the underlying mechanism to be
inferred from our data is that Lc3 synthase knockout
severely affected the functional integrity of B cells.
The expression of Lc3 synthase in splenic B cells is
interesting in light of the high level of activity with
gangliosides synthesized through GM3 and GM1 .
A rare species of hybrid ganglioside structures
containing extended lactosamine structures on a GM1 core was
identified previously in glycolipid extracts of splenic B
cells, but not T cells . These gangliosides were
shown to have the core structure GlcNAc(1-3)Gal(1-3)
GalNAc-R. It seems likely that Lc3 synthase is involved
in the synthesis of this GlcNAc(1-3) linkage. Although
the structural confirmation and significance of these
findings await further analysis, these results may reveal a
previously undescribed role for Lc3 synthase in
regulating synthesis of these unique glycolipids. The exact
functions of lacto-series glycolipids have remained
elusive, with the exception of a few specific processes.
The disruption of the Lc3 synthase gene in mice should
provide valuable insight into the functional involvement
of this glycolipid class in developmental and
We have not seen any report on obesity in ganglioside
knockout mice. The incidence of obesity was about 40%
in null mice, and it occurred only in male mutant mice.
There are genetic and immunologic data that strongly
suggest that type I diabetes may be associated with
autoimmune antibodies against gangliosides, mainly
disialo- or polysialo-gangliosides . Also, a growing body
of evidence indicates that mice without the capacity to
synthesize the a-series GM3 ganglioside display an
increased insulin sensitivity, and these mutant mice are
protected from high-fat-diet-induced insulin resistance
. Thus, gangliosides may be involved in occurrence
of obesity. In concordance with this hypothesis,
LastresBecker et al. recently reported the deletion of SCA2,
coding ataxin-2 protein and causing spinocerebellar
ataxin type 2 disorder, and found similar phenotypic
changes of obesity and reduced fertility .
Interestingly, they also found significant changes in the
expression level of gangliosides, mainly GM1, GD1a, GD1b,
and GT1b. They suggested that gangliosides are
characteristic constituents of membranes that are involved in
the insulin-signaling pathway. Although the gene they
knocked out was different from ours, the study did
indicate an interconnection between phenotypic changes of
obesity, reduced fertility, and ganglioside level.
Nevertheless, further experiments are necessary to better
define the possible relationship of our knockout models
Despite significant multiple phenotypic changes
observed here in some of the homozygote and
heterozygote mice, other mice were able to survive in
approximately normal health for more than one year. This
variability remains to be investigated, and there are
several possible explanations: First, Lc3 synthase, which
functions at the beginning of the lacto-neolacto-series
pathways, has an impact on the genes that affect the
formation of multiple molecules along the whole pathway
cascade. Thus, Lc3 synthase knockout may cause
multiple phenotypic changes. Second, because surface
ganglioside composition differspossibly individuallyin
quality and quantity, sensitivity to knockout may differ
individually. Third, the extent of ganglioside distribution
and functional overlapping or compensation may also
vary by individual experimental animal, especially in a
knockout model . All of these individual differences
may be contributing to phenotypic variations.
Furthermore, evidence has already been presented that there are
hybrid ganglioside molecules in the lacto-neolacto-series
pathways with the structure of N-acetylgalactosamine,
which has been defined as the major component of
the a- and b-series pathways , indicating the
possibility of functional overlapping within the different
ganglioside pathways. In addition, we also observed
some subtle neural phenotypic changes occurring in
Lc3 synthase-mutant mice, such as hind-limb
weakness, tremor, and seizure onset, demonstrating possible
It is known that gangliosides are poor antigens ,
and raising antibodies against gangliosides in wild-type
mice is difficult because of the poor antigenicity of the
gangliosides and because of immunological tolerance in
animals. We previously used 3-isoLM1 and 36-isoLD1
as targets to immunize several strains of mice and
encountered poor humoral immune responses,
producing only IgM antibodies , which confirmed
poor ganglioside antigenicity. On the other hand, in
addition to their overexpression on the surface of a wide
variety of tumors, gangliosides are involved as
autoantigens in the pathophysiology of human autoimmune
disorders in the peripheral nervous system and CNS
[3,38-41]. Endogenous and exogenous antiganglioside
antibodies contribute to induction of human
autoimmune nerve pathology. Nevertheless, anti-GD1a IgG
antibodies have been generated in GalNacT
(b-1,4-Nacetylgalactosaminyl transferase)-null mice [22,39]. Most
recently, we have successfully isolated specific
anti-3isoLM1 and anti-3,6-isoLD1 IgG clones from our
immunized Lc3 synthase knockout mice, 50% of the
clones being IgGs, in an attempt to use a low-toxicity,
high-efficiency fusion method via a Sendai virus
envelope. Further, after single cell cloning, one of the clones,
GMab-1 (IgG3 subclass), was established to specifically
recognize lacto-series gangliosides with high affinity.
Significantly, it was reactive against human glioma samples
in immunohistochemistry assays and thus demonstrated
potential as a good reagent for diagnosis and therapy
These novel results from the disruption of the B3gnt5
gene in mice demonstrate unequivocally that
lactoneolacto series gangliosides are essential to multiple
physiological functions, especially the control of
reproductive output, and spleen B-cell abnormality. However,
our results suggest the B3gnt5 gene is not essential for
mouse embryonic development. We also employed the
Lc3 synthase knockout model, lacking the
lactoneolacto-series, to successfully generate an IgG response
against 3-isoLM1 and 3,6-isoLD1 gangliosides. This led
to the subsequent first IgG antibody generation, which
was recently achieved by our group .
Generation of Lc3 synthase gene knockout mice
Two strategies were used to produce Lc3 synthase gene
B3gnt5 knockout models. For model 1, B3gnt5 was
knocked out by a conventional method. A 12.3-kb
region used to construct the targeting vector was first
subcloned from a positively identified BAC clone. The
region was designated such that part of intron 3 and all
of exon 4 of the gene were deleted and replaced with
the MC1NeoPolyA selection cassette. The targeting
vector was confirmed by restriction enzyme analysis after
each modification step and by DNA sequencing with
primers designed to read upstream of intron 3 and
downstream of exon 4 and the neo gene. This vector
was transfected into C57BL/6 ES cells. After
homologous recombination, the positive cells carrying a
targeted allele were screened by PCR and microinjected
into C57BL/6 blastocysts, and B3gnt5 knockout mice
were established through final germ-line transmission by
continuous breeding with the C57BL/6 strain.
For model 2, B3gnt5 was disrupted by a Cre-loxP
system. Since the coding region of this gene resides only
within exon 4, exon 4 of B3gnt5 was flanked by a pair of
loxP sites. In order to do this, we first designed specific
primers to amplify the B3gnt5 gene fragments from
129sv strain ES cells. A PCR product was used as a probe
to generate B3gnt5-positive BAC clones from the mouse
129sv strain by microarray screening. One genomic BAC
clone was used to construct the targeting vector
containing a 7.9-kb genomic DNA sequence with a floxed Lc3
synthase targeting allele. The resulting targeting clone,
pOSfrt-loxP-B3gnt5, confirmed by restriction enzyme
analysis and DNA sequencing, was transfected into the
129sv ES cells. Eighty clones were screened by Southern
blot to identify the recombinant cells carrying a floxed
B3gnt5 allele, which were then microinjected into
C57BL/6 blastocysts to generate the C57BL/6-129sv
chimeric mice. Chimeric males were then mated to C57BL/
6 females. Germ-line transmission of the floxed B3gnt5
allele was established by PCR screening of agouti
offspring. Mice bearing the floxed B3gnt5 allele were mated
to the general Cre-deleter mice (The Jackson Laboratory
mice database: B6.FVB-TgN[EIIa-Cre]), which resulted in
excision of the floxed B3gnt5 sequence.
Maintenance of knockout mice
All of the mice were kept in the Cancer Center Isolation
Facility at Duke. From one to five animals were
maintained in an individual cage on a regular dark/light cycle
with unrestricted access to food and water. The mice, of
three different genome types, were maintained through
intercross breeding between heterozygotes from the
same generation or through backcross breeding with
one of the parents. Knockout of the B3gnt5 allele was
confirmed by PCR of DNA prepared from tail biopsies
collected at weaning. To determine homozygotes of the
B3gnt5 allele, PCR products were purified and
sequenced to ensure their homogeneity.
All breeding was done in a C57BL/6 background.
Animals were maintained in accordance with National
Institutes of Health guidelines for the care and use of
laboratory animals and under the approval of the Duke
University Medical Center Institutional Animal Care
and Use Committee.
Genotyping of mice by PCR
A mouse tail kit (Qiagen, Valencia, CA) was used to
extract genomic DNA. For the conventional knockout
mouse model, genotyping was performed by PCR with
primers of 5exon 4 (5ATGAGACTGTTTGTTAGC)
and 3exon 4 (5GATTGTGGAAAGAATCAA). The
expected DNA product size for the wild type was 0.5 kb.
Also, neo gene primers with sequences as follows were
Neo1 R (5GCTACACAAGTGGCCTCTGGCCTCG
Neo7 R (5CTTCAGGCTATGAAACTGACACAT)
The size of the targeted DNA fragment was 1.9 kb.
For Cre-loxP knockout mice, PCR was performed with
the same exon 4 primer set plus Cre primers.
The size of the floxed B3gnt5 allele was 4.3 kb; that
for the knocked out B3gnt5 allele was 2.1 kb. PCR
cycling was based on the different primer sets.
Total RNA was extracted from frozen liver, spleen, and
brain of adult mice or from head and body of day E17
fetuses by using an RNeasy mini isolation kit (Qiagen)
as described by the manufacturer. About 5 l (1 g) of
total RNA was used for synthesis of first-strand cDNA
by using SuperScript II reverse transcriptase and a
random primer (Invitrogen, Carlsbad, CA). Subsequently,
1 g of cDNA was used for PCR with the same exon
4 primer set as was used for the above-described
genotyping. Mouse b-actin (primers from Invitrogen Biosource)
was used as an internal quality control to monitor RNA
recovery. Cycling was performed according to standard
procedure. Samples were run on a 1% agarose gel.
Perfusion, dissection, and histological analysis of
The anesthetized animals were perfused through a
cardiac tube with 4% paraformaldehyde in phosphate buffer
for approximately 2 h. Organs from the three mouse
genotypes, including heart, lung, spleen, cortex,
cerebellum, kidney, pancreas, stomach, small intestine, bladder,
uterus, testes, and skeletal muscle, were collected and
fixed in 4% paraformaldehyde for 12 h. The fixed tissues
were either protected in 30% sucrose for 2 days at 4C,
then flash-frozen in Tissue-Tek OCT compound (Sakura
Finetek USA Inc., Torrance, CA), or directly paraffin
embedded. Frozen or paraffin-embedded sections (5-10 M)
were cut and stained with H&E by using standard
Behavioral analysis of knockout offspring
The knockout offspring from heterozygous breeding
were matched either with wild types or with littermates
for behavioral studies, including appearance, weight,
survival rate, eating and drinking, and mating. Once any
special phenotype appeared, the mice were placed and
observed separately and then compared to their
littermates for at least 3 months.
Evaluation of reproductive performance
Heterozygous or homozygous knockout mice of similar
ages were bred from 5 to 9 months to assess the fertility
of mutants. Pregnancy was confirmed by observation of
positive plugs. The day when a plug appeared was
counted as day 0.5. The number of litters during the
breeding process, the litter sizes, and the number of
surviving pups and dead pups were recorded. For fetal
studies, pregnant females were dissected at days E15 to E18
while anesthetized. The fetuses per pregnancy were
counted and totals recorded. Fetuses were genotyped by
using the same mouse tail kit as was used for adult
The organs were removed by dissection from
anesthetized mice of the three different genotypes. Also, fetal
tissues were taken from the anesthetized dam, quickly
frozen in dry ice, and processed for the ganglioside
assay. The total ganglioside fraction was isolated
essentially as previously described . Quantitative
determination was made of lipid-bound sialic acid (total
gangliosides) by the resorcinol procedure, and the
distribution of the major ganglioside species was determined
by densitometric evaluation of a high-performance thin
layer chromatography (TLC) chromatogram after
resorcinol visualization . The lactoseries gangliosides
3-LM1, 3-isoLM1, and 3,6-isoLD1 were quantitated by
TLC-immunostaining using the specific monoclonal
antibodies LM1, TR4, and DMab 22, respectively .
Spleen immunophenotyping by fluorescence-activated
Flow cytometric analyses were performed on spleen cells
by following a standard protocol. Briefly, harvested cells
were washed twice with ice-cold phosphate-buffered
saline (PBS), and then, cell-surface nonspecific antibody
binding was blocked with 10% normal goat serum in
PBS (Sigma, St. Louis, MO). Blocked cells were mixed
with 10 g/ml of either anti-CD4 (clone L3T4), CD8
(53-6.7), CD19 (1D3), CD49b (DX5), or the appropriate
isotype-specific controls in ice-cold PBS-1% BSA
(Invitrogen). All antibody fluorescein conjugates were
acquired from BD Pharmingen (San Jose, CA). The cells
were incubated by rotating them in the dark for 60 min
at 4C, washed in PBS-1% BSA, and then resuspended
in 0.05% paraformaldehyde/PBS for acquisition on a
FACSCalibur flow cytometer (BD Biosciences, San Jose,
CA). The data were analyzed with BD CellQuest Pro
software (BD Biosciences) and represented as median
fluorescence intensity as compared to the appropriate
isotype control antibody.
Immunization of homozygous mice with gangliosides and
testing by ELISA
Homozygous knockout mice, age 2-4 months, were used
for immunization. Purified gangliosides, either 3-isoLM1
or 36-isoLD1, conjugated with Salmonella minnesota
were used as antigens. The conjugates were prepared as
follows. Gangliosides were dissolved in ethanol and acid
washed. Salmonella particles in water were added in the
weight ratio ganglioside-Salmonella particles 1:10. After
incubation for 1 h at +40C, the conjugated particles
were diluted with PBS and stored frozen until use. Then,
5 g of each antigen was injected subcutaneously 3-5
times at intervals of about 20 days. Sera were collected
and tested by ELISA for reactivity against the
gangliosides as optical density readings at 492 nm.
Pre-immunized mouse serum was used as the control baseline.
Data were expressed as mean standard deviation.
Results having a P value of < 0.05, or a P value of <
0.01 by the Student t test, chi square test, or binomial
test, were considered to be significant.
CGT: ceramide galactosyltransferase; CNS: central nervous system; CST:
cerebroside sulfotransferase; ES: embryonic stem; H&E: hematoxylin and eosin;
KO: knockout; PBS: phosphate-buffered saline; PCR: polymerase chain reaction;
RT-PCR: reverse transcription polymerase chain reaction; WT: wild type.
We thank Drs. Carol J. Wikstrand, Michael W. Graner, and Patrick J. Buckley
for helpful discussion. We also thank Shelley Davis and Ian Cumming of
Duke University Medical Center for their technical assistance and Mrs.
Birgitta Dellheden of the University of Gothenburg for the ganglioside
analyses. This investigation was supported by the following NIH grants:
Grant MO1 RR30, General Clinical Research Centers Program, National Center
for Research Resources; NINDS Grant N5 NS20023; NIH Merit Award R37 CA
011898; and NCI SPORE Grant 5P50 CA108786. We are also grateful for the
support received through a grant from the Pediatric Brain Tumor
CK initiated and directed the study, participated in the design of the study,
and made substantial revisions to the manuscript. JC participated in the
design of the study, drafted and wrote several revisions of the manuscript,
constructed the B3gnt5 targeting vector, screened ES cells for homologous
recombination, and was responsible for mouse breeding, phenotypic
characterization, and statistical analysis of all data. JEM and PF extracted and
analyzed GSL profiles in knockout mice and made significant revisions to the
manuscript. JL and CP performed the embryo isolation, mouse dissection,
and slide fixation. RM prepared the H&E slides and contributed the critical
interpretation of the histology. DB initiated and directed the study and
made significant revisions to the manuscript. All authors read and approved
the final manuscript.
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