Behavior Genetics, Vol. 36, No.

3, May 2006 (
2006)

DOI: 10.1007/s10519-006-9046-y

The Production of Transgenic Mice Expressing Human

Cystathionine Beta-Synthase to Study Down Syndrome
Christine Butler,1 Aaron J. Knox,1,2 Jerey Bowersox,1 Stacy Forbes,1
and David Patterson1,2,3
Received 6 Nov. 2005Final 17 Dec. 2005

Down syndrome (DS) is the most common genetic cause of signicant cognitive disability. We
hypothesize that by identifying metabolic alterations associated with cognitive impairment, it
may be possible to develop medical or dietary interventions to ameliorate cognitive disabilities
in persons with DS. Evidence suggests that one-carbon/transsulfuration (1C-TS) metabolism
is abnormal in persons with DS. Cystathionine beta-synthase (CBS) plays a critical role in this
metabolic system. The gene for CBS is on human chromosome 21, and there is evidence of
elevated CBS enzyme activity in tissues and cells from individuals with DS. To analyze the
possible role of CBS in Down syndrome, we have produced several lines of transgenic mice
expressing the human CBS gene. We describe the use of Florescence Situ Hybridization
(FISH) analysis to characterize the transgene insertion site for each line. Our initial expression
analysis of each transgenic line by RT-PCR shows that the tissue specicity of human CBS
mRNA levels in these mice may dier from the tissue specicity of mouse CBS mRNA levels
in the same animals. These mice will be invaluable for assessing the regulation of the CBS gene
and the role of CBS in cognition. They can also be used to develop therapies that target
abnormalities in 1C-TS metabolism to improve cognition in persons with DS.
KEY WORDS: Cognitive disability; cystathionine beta-synthase; down syndrome; gene expression;
transgenic mice.

risk of Alzheimers disease (AD) as they age. As the

life expectancy of persons with DS continues to
increase, this decline becomes an increasingly significant problem (Costa and Patterson, 2005).
It would be a major boon to persons with DS, to
their families, and to society as a whole if the cognitive
disability of persons with DS could be ameliorated.
Studies show that it is possible to improve memory
and learning in the Ts65Dn mouse model of DS
(Davisson et al., 1993). Granholm et al. (2002) demonstrated that estrogen treatment reverses cognitive
decline in 11- to 15-month-old female Ts65Dn mice.
We hypothesize that appropriate treatment with
drugs or dietary alterations could enhance the cognitive ability of persons with DS. If metabolic alterations can be associated with the phenotypes of DS, it
should be possible to devise treatments that normalize
metabolic activity with, for example, drugs or dietary

INTRODUCTION
Down syndrome (DS) is the most common genetic
cause of signicant cognitive disability, affecting
1/700 to 1/1000 live births (Roizen and Patterson,
2003). One of the most pervasive and signicant
features of DS is cognitive impairment. People with
DS not only have lifelong cognitive impairment, they
also suer from cognitive decline and an increased
1

The Eleanor Roosevelt Institute at the University of Denver,

1899 Gaylord St., Denver, CO, 80206, USA.
Human Medical Genetics Program, University of Colorado at
Denver and Health Sciences Center, Aurora, CO, USA.
To whom correspondence should be addressed at The Eleanor
Roosevelt Institute and the Department of Biological Sciences,
The University of Denver, 1899 Gaylord St., Denver, CO, 80206,
USA. Tel.: 303-336-5650; Fax: 303-333-8423; e-mail: dpatter2@
du.edu

429
0001-8244/06/0500-0429/0
2006 Springer Science+Business Media, Inc.

430

Butler, Knox, Bowersox, Forbes, and Patterson

(Peeters et al., 1986), a folate antagonist. Cells from
individuals with DS also show this sensitivity (Lejeune
et al., 1986). Further, Peeters et al (1995) showed that
supplementation with folic acid partially corrects in
vitro methotrexate toxicity, and hypothesized that
1C-TS metabolism might be involved in the pathogenesis of mental deciency in DS (Lejeune, 1990).
At least six genes encoding enzymes important
for 1C-TS metabolism are located on human chromosome 21, including the gene for CBS (Fig. 1,
Munke et al., 1988; Hattori et al., 2000). CBS catalyzes the condensation of serine and homocysteine to
form cystathionine. It plays a critical role in linking
the folate cycle and the methionine cycle and in regulating homocysteine levels. In addition, CBS can
convert cysteine to hydrogen sulde, which researchers are beginning to recognize as an important
neuromodulator in the brain (Eto and Kimura, 2002).
Mutations leading to deciency in CBS in humans can
lead to serious cognitive disability (Miles and Kraus,
2004; Mudd et al., 2001).
There is evidence that CBS protein levels and
enzyme activity are increased in persons with Down
syndrome (Chadefaux et al., 1985; Ichinohe et al.,

modications. We refer to such treatments here as

metabolic intervention. A systematic review of the
eect of therapeutic dietary supplements and drugs on
cognitive function in persons with DS (Salman, 2002)
concluded that of 11 trials with 373 randomized participants there was no reported cognitive enhancement in persons with DS. Overall, the design and
quality of the studies, however, made it impossible to
draw any rm conclusions. It is dicult to conduct
conclusive tests of hypotheses about metabolic intervention in humans because of the genetic heterogeneity of the human population, insuciently detailed
knowledge of which metabolic systems to focus on
and how they interrelate, the long lifespan of humans,
the inability to conduct concurrent invasive biochemical studies, the potential importance of duration
and timing of treatment, and diculty in controlling
experimental conditions with humans. Mouse models
make it possible to circumvent these diculties and to
continue the search for metabolic interventions to
improve cognition in persons with DS.
There is evidence of a link between 1C-TS
metabolism and DS. Children with DS show increased
sensitivity to the anti-leukemia drug methotrexate

L-gamma-glutamyl- cysteine

GSH

cysteine

H2S
+
CBS pyruvate

5-methylFH4

cystathionine

REFC

serine
HOMOCYSTEINE

(extracellular)

CBS

5-methylFH4

5-methylFH4glun

(intracellular)

5, 10-methyleneFH4glun
pyrimidines
5, 10-methenylFH4glun

betaine

FH4glun

choline

METHIONINE
CYCLE

FOLATE
CYCLE

FTCD

SAH

dimethylglycine
SAM

METHIONINE
10-formylFH4glun

Methylation
reactions

DNMT3L
N6AMT1?

HRMT1L1

GART
methylDNA
FGAR
+ FH4glun

FAICAR
+ FH4glun

methylprotein

purines

Fig. 1. A representation of 1-carbon/transsulfuration (1C-TS) metabolism. The genes in red are encoded on human chromosome 21.
CBS=cystathionine beta-synthase; REFC=reduced folate carrier; FTCD=formiminotetrahydrofolate cyclodeaminase; GART=glycineamide ribotide formyltransferase; HRMT1L1=arginine methyltransferase 1-like protein; DNMT3L=DNA methyltransferase 3-like enzyme;
N6AMT1?=putative N6 adenine methyltransferase 1; GSH=glutathione; SAH=S-adenosyl homocysteine; SAM = S-adenosyl methionine;
FH4glun=glutamyl-tetrahydrofolate; FGAR=formylglycinamide ribotide: FAICAR=formyl aminoimidazolecarboxamide ribotide.

Transgenic Mice Expressing Human Cystathionine Beta-Synthase to Study Down Syndrome

2005). Modulation of CBS activity in transgenic mice
containing human CBS cDNA under the control of
an inducible promoter showed that elevated CBS
activity can lower homocysteine levels (Wang et al.,
2004). Kamoun et al. (2003) showed that hydrogen
sulde levels may be elevated in individuals with DS.
If increased CBS activity decreases homocysteine
levels, perturbs the balance of 1C-TS metabolism and
leads to elevatedperhaps toxiclevels of hydrogen
sulde, these metabolic alterations might play a role
in the cognitive disability seen in Down syndrome
(Kamoun, 2001; Kamoun et al., 2003).
There are other possible mechanisms through
which the cognitive phenotype of DS could involve
CBS. For example, low levels of homocysteine might
result in a lack of active methyl groups and alter the
activity of proteins encoded by genes on chromosome
21, including DNMT3L, HRMT1L1, N6AMT1, and
GART, which utilize methyl groups. Al-Gazali et al.
(2001) described abnormal folate metabolism in a
child with DS and a neural tube defect. The chromosomally euploid mother had highly elevated
homocysteine and S-adenosyl homocysteine levels
and low methionine levels in plasma. The child had
low plasma levels of both homocysteine and methionine and also had a ve-fold increased level of cystathionine relative to levels observed in typical
children. This latter observation is consistent with
overexpression of CBS. Both the mother and child
had hypomethylation of lymphocyte DNA, which
would be expected from abnormal folate metabolism.
Additional research conrms and extends these
observations. Pogribna et al. (2001) examined homocysteine metabolism in 42 children with DS and 36
normal siblings. They found that the children with
DS had decreased plasma levels of homocysteine,
methionine, S-adenosyl homocysteine, and S-adenosyl methionine and elevated levels of cystathionine
and cysteine, as would be expected from elevated
CBS activity. A recent study of 131 individuals with
DS also showed generally low levels of homocysteine
and suggested that, within the DS group, individuals
with high homocysteine tended to have lower IQ
(Gueant et al., 2005).
Robert et al. (2003) demonstrated using immunolocalization and molecular techniques that CBS
mRNA and protein are indeed expressed during
mouse brain development and in adult brain regions,
namely the hippocampus and cerebellum, that are
altered in persons with Down syndrome. Using an
elegant immunohistochemical technique on mice in
which the CBS gene had been inactivated by targeted

431

mutagenesis (Watanabe et al., 1995) and relevant

controls (CBS wt and CBS heterozygotes), they
further showed that elevated homocysteine levels were
particularly striking in hippocampus and cerebellum.
CBS Transgenic Mice
On the basis of the results presented above, we
hypothesize that overexpression of CBS may play a
role in the cognitive disabilities faced by persons with
Down syndrome. Wang et al. (2004) reported production of transgenic mice expressing human CBS
from human CBS cDNA under the control of an
inducible promoter. These authors reported that
there were no observed dierences between control
and transgenic mice in growth rate, behavior or
reproductive ability. However, it is not clear from this
work what behavioral tests the authors used or how
long the mice were subjected to the conditions that
induced human CBS expression. In these mice, the
metallothionein promoter regulates expression of the
human CBS cDNA. This oers the advantage that
the expression of the human cDNA can be regulated
by simple experimental manipulations. However,
these mice cannot be used to investigate the regulation of genomic CBS by its endogenous promoter.
Moreover, the use of cDNA precludes study of the
role of alternative splicing of the CBS mRNA in its
regulation and in the activity of the CBS protein.
These regulatory mechanisms are most likely critical
for proper expression of CBS during mammalian
development and for proper control of CBS mRNA
and protein expression. A mouse model expressing
human CBS from genomic DNA containing the entire gene and its endogenous regulatory regions is
essential to determining the possible role of CBS in
the DS phenotype.
If CBS overexpression contributes to the cognitive disability seen in persons with DS, then treatments that decrease the expression of the human gene
would be desirable. Alternatively, one could develop
drugs that inhibit CBS enzyme activity. For either of
these studies, transgenic mice expressing the human
CBS gene under the control of its endogenous regulatory regions would be extremely helpful. This report
describes the production of such transgenic mice with
three different DNA constructs (Table I). BAC
KB2007G4 contains the CBS, U2AF1 (also known as
U2AF35), and alpha A crystallin (CRYAA) genes
(Hattori et al., 2000). Studies have shown that
CRYAA expression is largely restricted to the eye
(Sax et al., 1997), and expression of this transgene in

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Butler, Knox, Bowersox, Forbes, and Patterson

Table I. Characteristics of DNA Constructs used to Produce CBS Transgenic Mice

CLONE

GENE

KB2007G4
P102D1
60.4P102D1

GENE CONTENT

LOCATIONa

SIZE

CBS, U2AF1, CRYAA

CBS, U2AF1
CBS

43,333,04143,481,214
43,326,48243,402,781
43,333,52043,393,982
43,346,26543,374,378
43,386,13543,404,757
43,462,21043,465,982

148,173
76,299
60,462
28,113
18,662
3772

CBS
U2AF1
CRYAA
a

Locations are in base pairs as presented on NCBI Map Viewer, Homo sapiens build 35.1 master map, 2004.

the KB2007G4 mice was expected to be similarly

limited. P1 clone P102D1 contains the human CBS
gene and the human U2AF1 gene. The third DNA
construct is a 60.4 kbp DNA fragment isolated from
P102D1. This fragment contains only the CBS gene.
This report further describes the initial characterization of these transgenic mice, including Fluorescence
in Situ Hybridization (FISH) analysis of the transgene insertion site for each line (Korenberg et al.,
1999) and preliminary expression analysis by RTPCR. This initial characterization provides necessary
groundwork for a detailed examination of behavioral
and metabolic phenotypes.
MATERIALS AND METHODS

Transgenic mice were also prepared using BAC

clone KB2007G4 (Hattori et al., 2000) generously
supplied by Drs. N. Shimizu and J. Kudoh. DNA
was prepared from this BAC using a Nucleobond
A500 column (Clontech) and linearized by restriction digest with P1-Sce1 (NEB). This BAC contains
CBS, U2AF1, and CRYAA, as assessed by BLAST
analysis of the end sequences of the insert.
Transgenic Mouse Production
The Eleanor Roosevelt Institute Transgenic
Mouse Facility prepared the transgenic mice using
standard procedures (Hogan et al., 1994). KB2007G4,
P102D1 and 60.4P10D1 DNA was diluted in MTE to a
concentration of 23 ng/ll for injection.

DNA Isolation for Production of Transgenic Mice

Kraus et al. (1998) previously described the isolation and characterization of the CBS-containing
human P1 clone P102D1. The locations of the insert
ends of P102D1 were determined by end sequencing
and BLAST analysis of the human genome (Altschul
et al., 1990). This analysis showed that P102D1 contains CBS and also U2AF1, an RNA splicing factor
(Hattori et al., 2000: Pacheco et al., 2004). Because
P102D1 contains the U2AF1 gene in addition to the
CBS gene, Nhe1 restriction sites were identied in the
P102D1 insert that bracket a 60,452 bp segment
containing the complete human CBS gene but not the
5 end of the U2AF1 gene. P102D1 DNA was digested
at 37C for 2 h with Nhe1 (NEB), and the digested
DNA was separated by pulsed eld gel electrophoresis
(PFGE) on a 1% LMP agarose gel. A DNA fragment
of 60.4 kbp was isolated by excising the region of the
gel containing this fragment, melting in b-Agarase I
Reaction Buer (NEB), and digesting the agarose
with 25b-Agarase I (NEB). The DNA was precipitated from the digested agarose and resuspended in
MTE. The size and integrity of the recovered DNA
was veried by PFGE.

Genotyping
Using a PUREGENE Genomic DNA Purication Kit (Gentra), DNA was extracted from the tail
tissue of offspring of female mice that had been implanted with KB2007G4-, P102D1- or 60.4P102D1injected eggs. The DNA was then tested by PCR for
the presence of the CBS human transgene using human-specic primers. Founders were thus identied for each line and bred with wild-type mice to
generate transgenic colonies.
All descendants were tested for the presence of
the CBS human transgene at 34 weeks of age. DNA
was isolated from 2 mm tissue clipped from the end
of the tail and incubated overnight at 56C with
0.03 mg Proteinase K (Roche) in 200 ll PCR Buffer
with Nonionic Detergents (Induced Mutant
Resources: Tail DNA for PCR (No Organic Solvents). The Jackson Laboratory, 2005). The sample
was then heated to 95C for ten minutes to deactivate
the Proteinase K. The DNA was analyzed by PCR
(2 ll DNA per 20 ll reaction) using human-specic
primers that amplify a 264 bp segment of human
CBS exon 17 (hCBS_Ex17F1: 5-TGT TTC ACC

Transgenic Mice Expressing Human Cystathionine Beta-Synthase to Study Down Syndrome

AAG GAA ATA TTG AGA-3 and hCBS_Ex17R1:
5-AAA TGC CGC TGA TTG TTC AC-3; optimal
annealing temperature=60C).

433

spreads were captured with the Cytovision imaging

system (Applied Imaging Corp.).
RNA Isolation and cDNA Synthesis

Generation of Mice Diploid for the CBS Transgene

To determine if the insertion of each human
construct had inactivated an essential mouse gene,
breedings were established to generate mice diploid
for the CBS transgene. Transgenic males and females
were mated and their offspring genotyped. Pups in
which human CBS was detected by PCR (as above)
were then analyzed by Fluorescence in Situ Hybridization (FISH) to determine CBS transgene copy
number.

Fluorescence in Situ Hybridization (FISH)

To determine the number of integration sites of
the transgene and to verify that the CBS human
transgene had not integrated on mouse chromosome
17, metaphase spreads were prepared from transgenic
mouse spleens from each line and analyzed by FISH.
Each spleen was homogenized using a motorized
STIR-R homogenizer (TRI-R Instruments). The
spleen cells were pelleted by centrifugation, resuspended and plated 1:8 in 10020-mm cell culture
dishes (TPP) with 7 ml supplemented media (Metaphase Preparation from Mouse/Rat Spleen, Section
of Cancer Genomics, Genetics Branch, NCI National
Institutes of Health, 2005) and incubated at 37C and
5% CO2. After 4872 h, 85 ll 10 lg/ml colcemide
(Gibco) was added to each plate and the cells were
returned to the incubator for 15 min. Metaphase
spreads were prepared from these cells according to
the NIH Section of Cancer Genomics protocol
(2005).
A Nick Translation Kit (Vysis) was used to direct label P102D1 (human CBS) DNA and mouse
chromosome 17 subtelomere (17T) DNA with Spectrum Orange (Vysis) and Spectrum Green (Vysis),
respectively. Metaphase spreads were co-denatured
with 1 ll probe mixed with 4 ll Hybridization Buffer
(Vysis). Slides were hybridized overnight at 37C,
postwashed for 12 min in 0.4SSC at 731C and
counterstained with DAPI. Hybridized metaphase
spreads were visualized using an Olympus epiuorescence microscope and single, dual and triple band
pass lters (DAPI, Spectrum Orange and Spectrum
Green). A minimum of 10 metaphase spreads per
slide were analyzed, and representative metaphase

Brain, eye, heart, kidney, liver, lung, muscle and

spleen tissues (40160 mg of each tissue) were harvested from transgenic mice and wild-type (wt) littermates, ash frozen in liquid nitrogen and stored at
)80C for 28 weeks. Total RNA was then isolated
using the standard Tri Reagent protocol (Sigma).
RNA concentrations were calculated from the
absorbance (at 260 nm) measured with a DU-640
Spectrophotometer (Beckman).
Using 450675 ng RNA, 20-ll cDNA synthesis
reactions were prepared with the ThermoScript RTPCR System for First-Strand cDNA Synthesis (Invitrogen). For each mouse, the same amount of RNA
from each tissue was used for cDNA synthesis. After
the cDNA was synthesized, each sample was treated
with 1 ll Rnase H for 50 min at 37C.
RT-PCR
To verify cDNA integrity, each sample was tested by PCR (2 ll 1:4 dilution cDNA per 20 ll reaction) for the presence of mouse cytosolic superoxide
dismutase (SOD1) cDNA. cDNA from KB2007G4
transgenic mouse tissues was also tested for the
presence of human CRYAA cDNA, and cDNA from
the P102D1 and 60.P102D1 transgenic mouse tissues
was tested for the presence of mouse CRYAA cDNA.
All cDNA samples were then tested for both mouse
and human CBS cDNA. Species-specic primers
(Table II) were designed using published sequence
and exon data (Ensembl Genome Browser: http://
www.ensembl.org/index.html) and online Primer3
Software (Rozen et al., 2000).

RESULTS
FISH Analysis of Transgenic Mice
Transgenic mice were produced using three
DNA fragments (Table I) as described in Materials
and Methods. FISH analysis was used to detect the
integration of the human transgenes into mouse
chromosomes for each construct (Fig. 2). This
experiment shows that in each case the transgene has
integrated into a single site.
FISH analysis of P102D1 and KB2007G4
transgenic mice generated by mating two transgenic

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Butler, Knox, Bowersox, Forbes, and Patterson

Table II. Primer Sets used for RT-PCR

GENEa
mSod1
hCRYAA
mCryaa
hCBS
mCbs

PRIMER SEQUENCESb

OATc

AMPLICON SIZE

F: 5-AAG CAT GGC GAT GAA AGC-3

R: 5-TTC AGA CCA CAC AGG GAA TG-3
F: 5-CGT GGT ACC AAA GCT GAA CA-3
R: 5-CAC ATC GAG GAA GAT GAC GA-3
F: 5-GTA ATG CAC CAA CCA CAT GC-3
R: 5-CTG TGG CCA GCA TCC AAA-3
F: 5-TTA CAG AGT TTG AGC GGT GCT-3
R: 5-GGC TCC TTG GCT TCC TTA TC-3
F: 5-CTT TTC TCC CAT CCT TGC TG-3
R: 5-GAG ACC GGC ATT CTT TGA G-3

57C

491 bp

60C

249 bp

57C

322 bp

60C

217 bp

55C

404 bp

m=mouse; h=human; SOD1=cytosolic superoxide dismutase; CRYAA=alpha A crystallin; CBS=cystathionine beta-synthase.

F=forward; R=reverse.
c
Optimal Annealing Temperature.
b

Fig. 2. FISH analysis of metaphase chromosomal spreads from CBS transgenic mice. P102D1 DNA (red) and T17 DNA (green) were used to
detect the human CBS transgene and the subtelomeric region of mouse chromosome 17 respectively. (a) 60.4P102D1 transgenic mouse
chromosomes hybridized with both probes simultaneously. The single copy of the CBS transgene is not in mouse chromosome 17.
(b) KB2007G4 transgenic mouse chromosomes hybridized with both probes simultaneously. The CBS transgene appears in both copies of the
same mouse chromosome. The transgene is not in mouse chromosome 17. (c) P102D1 transgenic mouse chromosomes hybridized with
P102D1 probe. CBS transgene appears in both copies of the same mouse chromosome.

Transgenic Mice Expressing Human Cystathionine Beta-Synthase to Study Down Syndrome

parents further shows that some transgenic offspring
are homozygous for the transgene. (Fig. 2). So far,
mice homozygous for the transgene have not been
produced from the 60.4P102D1 line.
Simultaneous FISH analysis with a human CBS
probe and a DNA probe for the mouse chromosome
17 telomere (Korenberg et al., 1999) demonstrates
that the transgene is not on mouse chromosome 17 in
the KB2007G4 or 60.4P102D1 lines (Fig. 2).
Expression of the Human CBS Gene in the Transgenic
Mouse Lines
Tissues from each transgenic mouse line were
then analyzed by RT-PCR. Figure 3 shows the result
of this experiment. All RNA preparations were tested
for expression of mouse cytosolic superoxide dismutase (SOD1), and all show robust expression of this
gene (with the exception of spleen RNA from the
P102D1 transgenic mouse), indicating that the RNA
was of high quality. In addition, expression of mouse
or human alpha A crystallin (CRYAA) was examined
in each line. Mice made with the KB2007G4 BAC
were analyzed for expression of human CRYAA,
while the other two transgenic lines were analyzed for
expression of mouse CRYAA. As expected, robust
CRYAA expression was detected only in eye RNA.
The same RNA preparations were then analyzed
for expression of the human and mouse CBS genes.
In each case, a single RNA preparation was made for
each tissue, and the expression levels of mouse and
human CBS were measured using species-specic
CBS primers (Table II). All three transgenic mouse
lines express the human and mouse CBS genes.
Interestingly, in terms of tissue specicity, expression
of the human CBS transgene diers from expression
of the endogenous mouse gene in transgenic mice
made with each construct. Although these results are
not quantitative, the data suggest that human CBS is
more intensely expressed in mouse brain as compared, for example, to kidney and liver, while mouse
CBS is most highly expressed in liver.
DISCUSSION
The results presented here describe the initial
characterization of transgenic mice expressing the
human CBS gene. Three different constructs were
used to produce these mice, two of which, KB2007G4
and P102D1, contain other genes. For the P102D1
and KB2007G4 transgenic lines, it is possible to breed
mice that are homozygous for the transgene (Fig. 2).

435

This result demonstrates that the integration of the

human CBS transgene did not inactivate an essential
mouse gene in either line. Therefore, any changes in
these mice are most likely due to expression of the
transgene(s). Moreover, because the transgene did
not integrate on chromosome 17, the location of the
mouse CBS gene, these mice can be crossed with mice
in which the mouse CBS gene has been inactivated
(Watanabe et al., 1995) for future experiments.
Qualitative assessment of the tissue-specic pattern of expression of both the mouse and human CBS
genes in the same mice demonstrates that the human
CBS gene is expressed from each construct. Data on
the tissue specicity of human or mouse CBS gene
expression as assessed by CBS mRNA levels is very
limited. Nonetheless, it is possible to make some
comparisons of the results presented here with published data. The expression of mouse CBS in mouse
tissues observed in this study is generally consistent
with published data on the expression of human CBS
in human tissues (Bao et al., 1998; Quere et al.,
1999). That is, there is high expression in the liver,
lower levels of expression in brain and kidney, and
less expression in lung. The tissue specicity of human CBS expression in the three transgenic mouse
lines diers from the pattern of CBS expression in
human tissues and from expression of the endogenous mouse gene (Bao et al., 1998; Quere et al.,
1999). Specically, expression of the human CBS
transgene appears to be higher in mouse brain and
more ubiquitous than expression of the mouse gene.
There are several possible explanations for the
unusual pattern of human CBS expression observed
in these transgenic mice. First, these qualitative results may not accurately represent relative levels of
mouse and human CBS mRNA in different tissues.
Given the robustness and consistency of the qualitative PCR results with three different strains of
transgenic mice made with three different CBS-containing constructs, this seems unlikely. Second, there
could have been some experimental error in the RNA
preparations. This too is unlikely, since the same
RNA preparations were examined for expression of
both the human and the mouse genes, and since the
general pattern of expression of human and mouse
CBS is the same for each construct. In addition, the
expression of mouse soluble superoxide dismutase
(SOD1) was assessed as a control in every RNA
preparation (Fig. 3, A3, B3, and C3), and this gene
consistently showed robust expression, even in RNA
samples that showed low expression of one or both
CBS genes. Third, it is possible that the DNA clones

436

Butler, Knox, Bowersox, Forbes, and Patterson

Fig. 3. RT-PCR analysis of human and mouse CBS expression in mouse tissues from three dierent transgenic lines. B=Brain; E=Eyeball;
H=Heart; K=Kidney; Li=Liver; Lu=Lung; M=Muscle; S=Spleen. (A1), (B1) and (C1) show human CBS expression patterns in
transgenic mice from lines KB2007G4, P102D1 and 60.4P102D1, respectively. (A2), (B2) and (C2) show mouse CBS expression patterns in the
same three lines. The KB2007G4 and P102D1 mice each contained two copies of the human CBS transgene. The 60.4P102D1 transgenic
mouse had one copy. The primer set used to detect human CBS in this experiment amplied human liver cDNA (OriGene) and did not detect
human CBS expression in any non-transgenic control mouse tissues (data not shown). (A3), (B3) and (C3) show mouse SOD1 expression in all
tissues. (A3) also shows expression of human CRYAA in transgenic KB2007G4 eye. (B3) and (C3) show expression of mouse CRYAA in
transgenic P102D1 and 60.4P102D1 eye. The same RNA sample for each tissue was tested for the presence of each gene.

were rearranged when they integrated into the mouse

genome. If this occurred, the regulatory regions of the
transgenic DNA may have been rearranged in such a
way as to alter their function. It is unlikely, however,
that rearrangements leading to similar eects on
expression would have arisen in all three constructs
used to make the transgenic mice.
The most likely possibility is that the human
CBS regulatory regions respond differently in mouse
tissues than they do in human tissues and differently
than the mouse CBS gene regulatory regions respond
in mouse tissues. Kraus et al. (1998) identied two
major promoter regions of the human CBS gene: the
)1a and )1b promoters. Transcription factors Sp1
and Sp3 play a role in regulation of these CBS promoters (Ge et al., 2002; MacLean et al., 2004).
Importantly, MacLean et al. (2004) reported that
Kruppel-like (KLF) factors, which have limited tissue
distribution, can repress expression of the human
CBS promoters. As these authors point out, however,
regulation of CBS expression likely involves other
factors as well. However, only 5 kbp of the 5 region
was analyzed in that work. It may be that tissue
specicity of expression involves regions further from
the 5 end of the gene. In our case, the KB2007G4
clone contains over 100 kbp, the P102D1 clone over
28 kbp, and the 60.4P102D1 clone over 19 kbp 5 of
the CBS gene (Table I). Since the transgenic mice
made with KB2007G4 express the human CRYAA
gene (Fig. 3, A3), regions more than 90 kbp 5 of the

CBS gene are apparently present in these animals

(Table I). Recently, Deutsch et al. (2005) assessed
evidence for regulation of human CBS expression by
regions of the genome on other chromosomes, or
distant from CBS on chromosome 21. They found no
evidence for this type of regulation of CBS expression. This nding supports the hypothesis that the
expression of CBS is regulated by DNA immediately
5 of the coding region. The mice described here will
be useful for assessing the tissue specicity of
expression of both human and mouse CBS, and
comparing animals from different transgenic lines
may reveal whether there are additional 5 regulatory
regions farther away than the 5 kbp previously analyzed. Mice made with KB2007G4 and P102D1 will
be useful for assessing the specicity of expression of
the human U2AF1 and CRYAA genes as well.
Interestingly, the tissue specicity of a second
critical gene in 1C-TS metabolism, the reduced folate
carrier (REFC, Fig. 1) has recently been assessed in
human and in mouse (Liu et al., 2005; Whetstine
et al., 2002). While dierent methods were used in
these two manuscripts, the results suggest that tissue
specicity of expression of the REFC gene may be
dierent in mouse and human.
The mice described here were produced with the
aim of studying the role of an extra copy of the CBS
gene in a mouse model of Down syndrome. Now that
the mice have been shown to express human CBS, a
detailed behavioral and metabolic characterization of

Transgenic Mice Expressing Human Cystathionine Beta-Synthase to Study Down Syndrome

these mice is warranted, as are studies on the in vivo
regulation of CBS in these animals. In particular, it
will be important to quantitate the different human
and mouse CBS mRNA levels in tissues from these
mice, and to determine the reason for differences in
the expression patterns. These mice will be invaluable
for developing ways to specically inhibit transcription of the human CBS gene. Moreover, since the
mice presumably produce human CBS protein, they
will be very useful for developing effective in vivo
inhibitors of the human CBS protein. Analysis of
homocysteine and hydrogen sulde levels in tissues
and plasma from each line will also provide important insights into the possible effects of altered CBS
levels on 1C-TS metabolism.
The data presented here, in conjunction with
published data showing that tissue specicity of
expression of a second critical gene in the 1C-TS
pathway, the reduced folate carrier (REFC), is different in mouse and human, raise the possibility that
1C-TS metabolism may be different in mouse and
human (Liu et al., 2005; Whetstine et al., 2002). This
would mean that care must be taken in extrapolating
from studies of 1C-TS metabolism in mice to the
human situation.
An important question remains as to whether the
mRNA expression patterns accurately reect the CBS
protein levels in the various animals and tissues. It
will also be important to determine whether it is
possible to produce mice expressing only the human
form of CBS controlled by it own promoter regions
by breeding the transgenic mice with mice in which
the mouse CBS gene has been inactivated by targeted
mutagenesis (Watanabe et al., 1995). If these mice
can be produced, they will have many uses, including
the analysis of the human CBS gene and protein in an
animal model and the development of compounds
that can modulate the activity or expression of human CBS. It may be that a complete analysis of the
role of CBS in Down syndrome will require additional mice, for example, mice in which the human
coding region is under the control of the mouse regulatory regions. In any case, in depth analysis of the
phenotype of these mice should provide valuable insight into the role of 1C-TS metabolism in cognition.

ACKNOWLEDGMENTS
This work was funded by a grant from the
Fondation Jerome Lejeune (DP) and a grant from the
National Institute of Child Health and Human

437

Development (NICHD) (DP). The authors thank

Drs. Jan Kraus and Jana Oliveriusova (University of
Colorado at Denver and Health Sciences Center) for
assistance in DNA preparation for FISH and Jean
Smith and Lynne Meltesen (Colorado Genetics
Laboratory, Department of Pathology, University of
Colorado at Denver and Health Sciences Center) for
the FISH analysis. This manuscript is dedicated to
the memory of Dr. Linda Crnic, a dear and valued
colleague and friend.

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