Selection of effective antisense oligodeoxynucleotides with a green
fluorescent protein-based assay
Discovery of selective and potent inhibitors of glutathione
S
-transferase Mu expression
Peter A. C. ¢t Hoen
1,2
, Bram-Sieben Rosema
1
, Jan N. M. Commandeur
2
, Nico P. E. Vermeulen
2
,
Muthiah Manoharan
3
, Theo J. C. van Berkel
1
, Eric A. L. Biessen
1
and Martin K. Bijsterbosch
1
1
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, the Netherlands;
2
Division of Molecular Toxicology,
Leiden/Amsterdam Center for Drug Research, the Netherlands;
3
ISIS Pharmaceuticals, Carlsbad, California, USA
Antisense oligodeoxynucleotides (AS-ODNs) are frequently
used for the down-regulation of protein expression. Because

Keywords: antisense oligodeoxynucleotide; carcinogenesis;
genetic polymorphism; glutathione S-transferase; green
fluorescent protein.
Antisense oligodeoxynucleotides (AS-ODNs) are frequently
used for the down-regulation of gene expression, both
in vitro and in vivo [1–4]. Due to the low stability of
phosphodiester ODNs (PO-ODNs) in biological systems,
more stable oligonucleotide analogues with a variety of
chemical modifications have been developed [5,6]. ODNs
with a phosphorothioate-modified backbone (PS-ODNs)
are the most commonly used AS-ODNs. As AS-ODNs act
via Watson–Crick base pairing with their target mRNAs,
the nucleotide sequence of the target gene is in principle
sufficient information for the design of AS-ODNs. It
appears, however, that not all AS-ODNs are potent
inhibitors of protein expression. In studies where large sets
of PS-ODNs, directed against a single target gene, were
tested for their ability to down-regulate their target mRNA
and protein in cell culture [7,8], only 5–10% of the sequences
tested appeared to be effective. Thus there is a need for rapid
and accurate screening assays for the selection of effective
and specifically acting AS-ODNs.
Screening for effective antisense sequences is usually
performed in cell-free systems or in cell culture. Several cell-
free assay systems have been described [9]. These assays are
fast, but not always reliable predictors for activity in
biological systems. Use of differentiated cells generates more
relevant information on the effectiveness of AS-ODNs in
physiological systems. However, cellular assays are fre-
quently hampered by low or irreproducible transfection of

With this assay, we determined the effectiveness of a set of
antisense PS-ODNs directed against the glutathione
S-transferase Mu1 (GSTM1) and Mu2 (GSTM2) isoforms
of the rat. GSTs play an important role in the detoxification
of DNA- and/or protein-reactive compounds by catalyzing
the conjugation of electrophilic groups with the tripeptide
glutathione [10]. Approximately 50% of the Caucasian
population is deficient for GSTM1, the human orthologue
of rat GSTM1 and GSTM2 [11]. Meta-analyses of epide-
miological studies reveal that this deficiency is associated
with an increased risk of lung and colorectal cancer,
especially when the GSTM-null genotype is combined with
high-inducibility of cytochrome P450 1A1 [12–18]. It would,
however, be very important to demonstrate directly the
effect of differences in GSTM expression levels on the
prevalence of cancer biomarkers in in vitro andinanimal
models. As GSTM knock-out mice are still unavailable,
temporal modulation of the expression of GSTM isoforms
by AS-ODNs in relevant in vitro and in vivo models is an
attractive possibility. In the current paper, several target-
specific AS-ODNs are selected from a set of 15 PS-ODNs.
These ODNs selectively inhibit the expression of GSTM1
and/or GSTM2 and can be used to study the influence of
reduced GSTM expression on the detoxification of xeno-
biotics and protection against chemical-induced carcino-
genesis.
MATERIALS AND METHODS
Materials
PCR primers were from Eurogentec, Seraing, Belgium.
PS-ODNs were synthesized according to standard phos-

hanging ends. The PCR products were cloned into the
EcoRI- and BamHI-digested pEGFP-C1 plasmid to gener-
ate the C-terminal fusion constructs pEGFP-M1 and
pEGFP-M2. Sequencing of the plasmids confirmed the
in-frame ligation of the GSTM cDNAs and the absence of
any PCR-induced mistakes in the inserts.
TRITC-labelling of ODN
A 24-mer PO-ODN, provided with three PS-linkages at the
5¢-end and a 3¢-end primary amino group (sequence:
T*A*A*GCTGTCCCGGGGTCTACGGCC), was label-
led with TRITC by incubating 15 nmol ODN in 500 lL
0.1
M
Na-carbonate buffer (pH 9.0) with 10 molar equiv-
alents of TRITC (dissolved in dimethylformamide at
2mgÆmL
)1
). The mixture was incubated overnight with
shaking at room temperature. The TRITC-labelled ODN
was separated from unreacted TRITC by gel filtration on a
Sephadex G-25 column (20 · 0.4 cm), eluted with water.
The TRITC-ODN was precipitated from the eluent by
adding 0.01 vols 1
M
MgCl
2
,0.1vols3
M
NaAc pH 5.2,
and 3 vols cold ethanol. The precipitate was formed by

pEGFP-C1.
Primer Sequence
GSTM1-forA 5¢-AATTCCATGCCTATGATACTGGGAT-3¢
GSTM1-forB 5¢- CCATGCCTATGATACTGGGAT-3¢
GSTM1-revA 5¢- CTAAAGATGAGACAGGCCTGG-3¢
GSTM1-revB 5¢-GATCCTAAAGATGAGACAGGCCTGG-3¢
GSTM2-forA 5¢-AATTCGATGCCTATGACACTGGGTTAC-3¢
GSTM2-forB 5¢- CGATGCCTATGACACTGGGTTAC-3¢
GSTM2-revA 5¢- CGTGGTTCACACTTTATTGCAAATC-3¢
GSTM2-revB 5¢-GATCCGTGGTTCACACTTTATTGCAAATC-3¢
Ó FEBS 2002 EGFP-based selection of antisense sequences (Eur. J. Biochem. 269) 2575
at a density of 1 · 10
5
cells/well,whichresultedincultures
that were approximately 50% confluent at the day of
transfection. For cotransfections of plasmid and ODN, the
appropriate amount of plasmid [diluted to a concentration
of 0.2 lgÆlL
)1
in HBS (0.15
M
NaCl in 20 m
M
Hepes,
pH 7.4)] was mixed with the appropriate amount of ODN
(diluted to a concentration of 0.2 lgÆlL
)1
in HBS). Subse-
quently, a transfection mixture was prepared by slowly
adding the plasmid/ODN mixture to a solution of DOTAP

analysis by gating of propidium iodide-positive cells.
GST activity assay
COS-7 cells were transfected with either pEGFP, pEGFP-
M1 or pEGFP-M2 as described above. Then, the cells were
washed twice with NaCl/P
i
and lysed in 300 lL10m
M
sodium phosphate buffer (pH 7.4) containing 2 m
M
dithio-
threitol, 1 m
M
EDTA, and 50 l
M
PMSF. The lysates were
homogenized by short sonication. Total GST activity was
analysed in a CDNB conjugation assay, essentially as
described before [20]. The assay makes use of the GST-
catalyzed addition of GSH to CDNB. The CDNB–GSH
conjugate formed can be measured spectrophotometrically.
To this end, 50 lL of protein lysate ( 10 lgprotein,
concentration determined with the Bradford protein assay
[21]) was incubated with 150 lL of a solution of 1.67 m
M
CDNB in 0.1
M
potassium phosphate buffer pH 6.5. Lysis
buffer, instead of lysate, was used as a blank. The reaction
was started by the addition of 50 lL5m

, 5% w/v milkpowder, 1% w/v BSA, 0.25%
v/v Tween-20. Then, the membrane was incubated for 1 h at
room temperature with the primary anti-EGFP antibody
(100 · diluted in blocking buffer without milk powder,
containing 0.5% v/v Tween-20). The membrane was
washed 10 times with NaCl/P
i
containing 0.02% v/v
Tween-20, and incubated with a horseradish peroxidase-
conjugated donkey antirabbit IgG (10 · dilutedin10·
diluted blocking buffer). EGFP was detected by an
enhanced chemiluminescence assay, according to the
manufacturer’s protocol.
Statistical analysis
Data were analysed statistically for significance with a one
or two sample student t-test.
GRAPHPAD INSTAT
Software
version 3.00, GraphPad Software Inc. (San Diego, CA,
USA), was used for this purpose.
RESULTS
Cloning of EGFP–GSTM fusion constructs
Cellular screening of antisense sequences for their potential
to inhibit gene expression is often complicated by irrepro-
ducible transfection procedures and lack of good quantita-
tive assays for monitoring of gene expression. To
circumvent these problems, we developed a screening assay,
based on fusion proteins of the target protein with EGFP,
that enables accurate determination of the effects of
AS-ODNs by flow cytometry. C-terminal fusion constructs

the fusion proteins was  50 kDa, as determined by
Western blotting with an EGFP-specific primary antibody
(Fig. 1B). This value is in close agreement with the expected
size, calculated by summation of the molecular weights of
EGFP (25 kDa) and GSTM (27 kDa).
Colocalization of ODN and pEGFP
For proper evaluation of antisense effects, it is important
that the AS-ODNs and the EGFP-expressing plasmids are
transfected into the same cells. This was accomplished by
the cotransfection of plasmid and AS-ODN. By FACS
analysis, it was shown that after cotransfection of COS-7
cells with a fluorescently labelled ODN and pEGFP, ODN
and plasmid colocalized in the same target cells as > 90% of
the EGFP-positive cells were also positive for the TRITC-
labelled ODN (Fig. 2). The observations suggest that the
uptake of ODN is far more efficient than the uptake of the
EGFP plasmid, because almost all cells were positive for
TRITC-labelled ODNs, whereas only  33% of the cells
were expressing EGFP.
Screening of ODNs for their antisense activity
To identify AS-ODNs that are potent and sequence-specific
inhibitors of GSTM1 and/or GSTM2 expression, 15 PS-
ODNs were screened for their ability to inhibit EGFP–
GSTM fusion protein expression. The ODNs were targeted
against different regions in the mRNA of GSTM1 and
GSTM2 (Table 2). Some of the ODNs (i.e. AS-1, AS-6,
AS-7, AS-8, AS-12 and AS-15) were designed to inhibit
selectively GSTM1 expression, whereas others (i.e. AS-5,
AS-10, AS-11, AS-13 and AS-14) were designed to inhibit
selectively GSTM2 expression. A third group of ODNs (i.e.

COS-7 cells were transfected with pEGFP-M1, pEGFP-M2, or
pEGFP (0.5 lg DNA per well). After a further 18 h of culture, the cells
were lysed. (A) Total GST activity in 50 lL of protein lysate was
measured by following CDNB–GSH conjugate formation over time.
The increase in absorption at 340 nm was recorded with lysis buffer as
a blank. The GST activity is expressed as a percentage of the activity in
pEGFP-transfected cells (0.46 DA
340
Æmin
)1
Æmg protein
)1
). Means of
12 determinations in three separate experiments ± SEM are shown.
**P < 0.0001 (unpaired student t-test). (B) A Western blot was per-
formed on 5 lg protein lysate of pEGFP-M1 (lane 1), pEGFP-M2
(lane 2) and pEGFP (lane 3) transfected cells. The samples were
denatured, and separated by SDS/15% PAGE, together with a
Bio-Rad prestained kaleidoscope protein marker. Subsequently,
proteins were blotted onto a nitrocellulose membrane, and the blot was
incubated consecutively with a rabbit anti-EGFP antibody and a
peroxidase-labelled goat anti-rabbit secondary antibody. EGFP-
containing proteins were visualized with enhanced chemiluminescence.
The positions and molecular weights of the marker proteins are indi-
cated in the left margin. In the right margin, the estimated sizes of the
protein bands are shown.
Ó FEBS 2002 EGFP-based selection of antisense sequences (Eur. J. Biochem. 269) 2577
AS-ODN did not have any effect on the expression of
EGFP, EGFP–M1 or EGFP–M2, indicating that cotrans-
fection of PS-ODNs per se does not influence EGFP

both GSTM1 and GSTM2, inhibited the expression of the
EGFP–GSTM isoforms by  95% (AS-2) and  80%
(AS-3), while expression of the control EGFP was inhibited
by 45 ± 12% and 18 ± 13%, respectively (Fig. 3C). AS-9
demonstrated severe nonspecific effects on EGFP expres-
sion, as the expression of EGFP, alone or in a fusion
construct, was inhibited by > 95%. The effects on EGFP
expression were not due to sequence-specific hybridization
because a significant homology with the sequence of EGFP
was not found. AS-4 showed less severe, but significant,
nonspecific effects on EGFP expression. Surprisingly, the
AS-GFP, which was reported to down-regulate EGFP
expression [23], did not have any effect on the expression of
either of the EGFP proteins at the tested concentration of
0.5 l
M
.
Fig. 2. FACS analysis of COS-7 cells
cotransfected with TRITC-ODN and pEGFP.
Untransfected COS-7 cells (A), cells trans-
fected with 0.2 l
M
TRITC-ODN (B), cells
transfected with 0.5 lg pEGFP (C), and cells
transfected with 0.2 l
M
TRITC-ODN and
0.5 lg pEGFP (D) were analysed by flow
cytometry for EGFP expression (FL-1, x-axis)
and TRITC-ODN uptake (FL-3, y-axis).

against both GSTM isoforms, and AS-6, directed at
GSTM1, potently inhibited the expression of their respect-
ive targets with IC
50
values slightly above 0.1 l
M
. At
0.1 l
M
, the inhibition was specific. However, at the highest
concentration tested also the expression of EGFP was
affected, indicating that sequence-specific antisense effects
occur at lower concentrations than non-target-specific
effects. AS-9 was the most potent inhibitor of both
EGFP–M1 and EGFP–M2 expression with estimated
IC
50
values < 0.1 l
M
. However, the EGFP expression
was also inhibited, although to a slightly lesser extent. AS-1,
targeted to GSTM1, was another nonspecific inhibitor of
EGFP expression as the IC
50
values for inhibition of EGFP
expression and of EGFP–M1 were both in the same range.
Analysis of the effect of AS-ODNs on GST activity
To examine whether the inhibitory effects of the AS-ODNs
on EGFP–GSTM fusion protein expression were associated
with a decrease in GST activity, we determined the effect of

present study a novel cellular screening assay for the
selection of effective AS-ODNs with a sequence-specific
Table 2. Antisense ODN sequences.
Target site
b
Mismatch
(number of bases)
c
Name Sequence
a
Region
b
GSTM1 GSTM2
AS-ctrl TGAGAGCTGAAAGCAGGTCCAT Unrelated – –
AS-GFP G*A*GCTGCACGCTGCCG*T*C GFP–CDS – –
AS-1 GGCGG
ATCGGGTGTGTCAGC CDS 36–55 – 5
AS-2 CCACTGGCTTCTGTCATAGT CDS 119–138 119–138 0
AS-3 GAAGTCCAGGCCCAGTTTGA CDS 152–171 152–171 0
AS-4 TCAATTAAGTAGGGCAGATT CDS 175–194 175–194 0
AS-5 TCTCCA
AAACGTCCACACGA CDS – 285–304 4
AS-6 ACAAAGCATGATGAGCTGCA CDS 326–345 – 8
AS-7 GAGTA
GAGCTTCATCTTCTC CDS 397–426 – 1
AS-8
ACTGGTCAAGAATGTCATAA CDS 480–499 – 7
AS-9 CAGGTTTGGGAAGGCGTCCA CDS 524–543 524–543 0
AS-10 CAGGCCCTC
AAACCGAGCCA CDS – 554–573 3

antisense effectiveness. Our experiments in which an EGFP-
containing plasmid was cotransfected with fluorescently
labelled ODNs, suggest that all EGFP-positive cells had
taken up ODNs. Therefore, in the present assay the antisense
effects are determined in the whole population of cells that
express the target gene. In other cellular assays, including a
luciferase reporter gene-based assay [24], antisense effects
may be underestimated because not all cells that express the
gene of interest are transfected with AS-ODNs. An EGFP-
based approach has been used previously for the selection of
ribozymes against the c-erbB-2 oncogene [25]. However, in
this earlier study the plasmid coding for the c-erb-B-2 EGFP
fusion protein, was cotransfected with a ribozyme expressing
plasmid and not with an exogenously added antisense
molecule. Cotransfection with the ribozyme-expressing
plasmid resulted in a reduction of EGFP expression to a
maximum of 70%, whereas we observed a > 90% reduction
with our most potent ODNs. Possibly, a significant part of
the c-erbB-2-EGFP transfected cells had not taken up a
ribozyme construct.
A C-terminal fusion construct and not an N-terminal
fusion construct, was used because AS-ODNs against the
3¢-untranslated region of the mRNA of the gene of interest,
which has been shown to be a favourable region for
antisense action [7], can only be tested in C-terminal fusion
constructs.
The newly developed screening assay was used for the
selection of effective AS-ODNs against rat GSTM1 and
GSTM2 out of a set of 15 PS-ODNs. Some ODNs were
designed to specifically inhibit either GSTM1 or GSTM2

effects on the expression of target-related control proteins,
which is easily accomplished in our screening assay.
Fig. 3. Effects of AS-ODNs on EGFP and EGFP–GSTM fusion
protein expression. COS-7 cells were transfected with 0.5 lg pEGFP
(open bars), pEGFP-M1 (hatched bars) or pEGFP-M2 (closed bars),
together with 0.5 l
M
of the indicated AS-ODNs. The ODNs were
directed against GSTM1 (A), GSTM2 (B), or both GSTM isoforms
(C). AS-ctrl is a control ODN without sequence homology with
GSTM or EGFP. AS-GFP is an AS-ODN against EGFP, taken from
[23]. At 22 h after transfection, cells were analysed for EGFP expres-
sion (FL-1) and propidium iodide uptake (FL-3) by flow cytometry.
The number of living (i.e. propidium iodide-negative), EGFP-positive
cells was counted and is expressed as the percentage of EGFP-positive
cells in cultures transfected with plasmid, but without AS-ODN.
Means of three independent experiments ± SEM are shown.
*P < 0.05; **P < 0.005 (one group student t-test compared to con-
trol without AS-ODN).
2580 P. A. ’t Hoen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Table 3. Effects of AS-ODNs on GST activity. COS-7 cells were transfected with 0.5 lg pEGFP, pEGFP-M1 or pEGFP-M2, together with 0.1 l
M
of the indicated AS-ODNs. AS-5 and AS-10 are directed against GSTM2; AS-6 is directed against GSTM1. At 22 h after transfection, GST activity
in the protein lysates was determined by assaying CDNB conjugation over time. Conjugation rates are expressed as percentages of EGFP controls
(column 2, 3 and 5), or percentages of the additional EGFP-M1-dependent (column 4) or EGFP-M2-dependent (column 6) GST activity,
calculated by subtraction of the endogenous GST activity, which was determined in pEGFP-transfected cultures. Means of 10–12 determinations in
three separate experiments ± SEM are shown. Statistical significance of the difference between AS-ODN-treated and untreated cultures are
indicated:
a
P < 0.005,

c
AS-6 99 ± 6 110 ± 4
b
5±2
d
146 ± 6
a
57 ± 6
d
AS-10 95 ± 5 244 ± 9
b
61 ± 4
e
125 ± 7
a
30 ± 6
e
Fig. 4. Concentration-dependent inhibition of
EGFP and EGFP–GSTM fusion protein
expression by AS-ODNs. COS-7 cells were
transfected with 0.5 lgofpEGFP(n), pEG-
FP-M1 (j)orpEGFP-M2(d), together with
the indicated concentrations of AS-1 (A),
AS-2 (B), AS-5 (C), AS-6 (D) or AS-9 (E). AS-
1 and AS-6 are directed against GSTM1, AS-5
is directed against GSTM2, whereas AS-2 and
AS-9 are complementary to both GSTM1 and
GSTM2. At 22 h after transfection, cells were
analysed for GFP expression (FL-1) and
propidium iodide uptake (FL-3) by flow

the ODNs were both found to be concentration-dependent.
The IC
50
values of the most potent, specifically acting
AS-ODNs were  0.2 l
M
. It should be noted that for most
AS-ODNs, with the exception of AS-5, the concentration
window where sequence-specific antisense effects were
observed, was narrow. This was also found in other studies
where PS-ODNs were used, and may be explained by the
relatively low affinity of PS-ODNs for their target mRNA
sequences together with the high incidence of nonantisense
effects [1,24]. It is therefore of highest importance to evaluate,
in each antisense study, the concentration–activity profile.
The nature of the nonspecific effects elicited by PS-ODNs
remains to be clarified. With the possible exception of AS-11,
which contained only five mismatches with respect to the
EGFP sequence, neither of the AS-ODNs against GSTM
showed significant sequence homology with EGFP. Thus,
the observed effects on EGFP expression are probably not
caused by partial hybridization of the AS-ODNs with the
EGFP mRNA. We cannot exclude that some of the
nonspecific AS-ODNs decrease the transfection efficiency
of the EGFP plasmids. However, from earlier studies it
became apparent that sequence-dependent variations in
cationic lipid-mediated transfection efficiencies were small,
unless homo-oligonucleotides, such as A
18
, were applied

or inhibit protein expression by nonantisense mechanisms.
The effectiveness of the selected AS-ODNs will be evaluated
further in rat hepatocytes and in vivo, potentially allowing
the study of the effect of decreased GSTM expression on the
toxicity and carcinogenicity of xenobiotics.
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