Received for publication, November 20, 2002, and in revised form, January 17, 2003
Successful gene-targeting reagents must be
functional under physiological conditions and must bind chromosomal
target sequences embedded in chromatin. Triple helix-forming
oligonucleotides (TFOs) recognize and bind specific sequences via the
major groove of duplex DNA and may have potential for gene targeting
in vivo. We have constructed chemically modified,
psoralen-linked TFOs that mediate site-specific mutagenesis of a
chromosomal gene in living cells. Here we show that targeting
efficiency is sensitive to the biology of the cell, specifically, cell
cycle status. Targeted mutagenesis was variable across the cycle with
the greatest activity in S phase. This was the result of differential
TFO binding as measured by cross-link formation. Targeted cross-linking
was low in quiescent cells but substantially enhanced in S phase cells with adducts in ~20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could
be recovered by direct screening of colonies chosen at random. These
results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to
reflect a combination of appropriate oligonucleotide chemistry and
manipulation of the cell biology.
 |
INTRODUCTION |
Effective gene-targeting reagents would have a broad application
as probes of chromatin structure and in a variety of genomic manipulations, e.g. gene knock-out, targeted gene conversion
and/or recombination, and gene therapy. Successful constructs must be functional in the nuclear environment of living cells. Furthermore, their intended targets must be accessible despite the packaging and
condensation of nuclear DNA by chromosomal proteins. One targeting strategy that has been of interest for many years is based on triple-helix forming oligonucleotides
(TFOs).1
A DNA triple helix can form when a TFO lies in the major groove of
intact duplex DNA (1-5). The most stable structures assemble on
polypurine:polypyrimidine sequences with hydrogen bonds formed between
the bases in the third strand and those in the purine strand of the
duplex. The purine or pyrimidine motif third strands may be involved in
triplex formation depending on the target sequence, and a binding code
for the design of the third strands has been defined (6). Although
conventional oligonucleotides have features that limit their activity
under physiological conditions, there is a variety of chemical
modifications that ameliorate these limitations (7). Thus, for
pyrimidine TFOs, the replacement of cytosine by 5-methylcytosine and
the use of 2'-O-methyl (2'-OMe)-modified sugars permit
stable triplex formation at physiological pH in vitro
(8-13). Other derivatives contribute to the bioactivity of TFOs of
both motifs as shown by us and others in recent publications (14-18).
Although chemical modifications can enhance TFO affinity and triplex
stability, they obviously cannot address the issue of access to
chromosomal targets inside living cells. In biochemical experiments, it
has been shown that sequences in nucleosomes are poor candidates for
triplex formation (19-22). Thus, in the cell, a TFO is likely to
confront the same issues of target accessibility described for sequence
specific regulatory proteins (23). Of course, reports of gene targeting
in vivo by TFOs imply, of necessity, some degree of target
access (14, 18, 24-26). In addition, TFO binding to chromosomal
targets in permeabilized cells has been shown (27, 28).
We have employed an Hprt knock-out assay to measure the
activity of TFOs linked to the DNA cross-linker psoralen (pso-TFOs). The assay reflects the binding of the target sequence by the TFO and,
following photoactivation of the psoralen, the generation of an
inactivating mutation as a result of cellular processing of the
cross-link. We have used this approach to measure the activity of TFOs
carrying sugar and base modifications and have shown that TFOs with
2'-O-aminoethyl (2'-AE) substitutions are bioactive (18). We
have now asked whether the biological state of the cell could influence
TFO activity. Here we show that the frequency of mutagenesis induced by
a pso-TFO differs in quiescent and S phase cells. The difference
reflects the levels of targeted cross-links in the two cell populations.
 |
MATERIALS AND METHODS |
Cells and Synchronization Protocol--
Chinese hamster ovary
(CHO) AA8 cells (ATCC) were grown in Dulbecco's modified Eagle medium
(Invitrogen) supplemented with 10% fetal calf serum and
penicillin and streptomycin. Prior to an experiment, the cells were
grown in HAT medium (10
4 M hypoxanthine,
5 × 10
5 M aminopterin,
10
5 M thymidine) for 1 week. The cells were
synchronized in G0/G1 by a variation of the
method described by Sawai et al. (29). Cells were plated at
subconfluent levels, and on the next day, the medium changed to
Dulbecco's modified Eagle medium with 2% fetal bovine serum and 2%
Me2SO. After 48 h, the cells were washed (~85% G0/G1 cells by fluorescence-activated
cell sorter analysis) and either electroporated or fed with complete
medium (for G1 phase experiments) or incubated with
complete medium containing 100 µM mimosine for 16 h
to block them in early S phase (~90% early S cells) (for complete
details on this procedure, see Ref. 30). After 16 h, the cells
were released from the mimosine block by feeding with Dulbecco's
modified Eagle medium, 10% fetal bovine serum.
TFO Electroporation, Psoralen Treatment, and Hprt Mutation
Assay--
Cells were suspended at 107/ml and mixed with
TFO at 5 µM. The cells were electroporated (Bio-Rad)
followed by incubation at room temperature for 3 h and a 3-min
exposure in a Rayonet chamber to UVA light at 1.8 J/cm2.
The electroporation conditions were chosen to minimize cell toxicity
(trypan blue staining showed ~95% viable cells after UVA treatment).
Cells treated with free psoralen were incubated with 5 µM
psoralen for 30 min followed by UVA treatment. The cells were passaged
and then exposed to thioguanine selection (17).
Restriction Resistance Analysis of Non-selected
Clones--
Following TFO electroporation, UVA treatment, and culture
for 3-5 days to permit mutagenesis, 100 cells were plated in 60-mm dishes in standard growth medium. Individual colonies were expanded, and the DNA was extracted followed by amplification of the I4E5 target
region and digestion of the PCR products with XbaI.
Cross-link Analysis--
After UVA exposure of cells treated
with the pso-TFO, the DNA was extracted and digested with
EcoRI. 10 µg in 10 µl of solution were then mixed
with 45 µl of 98% formamide and heated at 77 °C for 15 min. The
samples were electrophoresed in an agarose gel in neutral buffer and
transferred to a nylon membrane. The membrane was hybridized with a
2-kb EcoRI fragment containing the I4E5 target region and
adjacent sequence. The probe was labeled by random priming and
incubated overnight with a blank membrane in hybridization buffer to
remove radioactive species that bound nonspecifically. This step
greatly reduced nonspecific binding to the nylon. In other experiments,
following TFO/UVA treatment, genomic DNA was exhaustively digested with
EcoRI and XbaI, blotted, and hybridized. To
control for triplex formation resulting from the interaction of unbound
TFO and the target during DNA purification (31), we mixed AE-07 and
cells and then isolated genomic DNA and digested it. There was no
XbaI-resistant band.
 |
RESULTS |
The CHO I4E5 HPRT Target and pso-TFO--
The triplex
target sequence is in the fourth intron next to Exon 5 of the CHO
Hprt gene (Fig.
1a). The sequence consists of a 17-base polypurine:polypyrimidine sequence ending in a 5'-TA step,
which is appropriate for T-T interstrand cross-linking by psoralen
(32). The A is the first base of the AG splice acceptor sequence, and
point mutations at this site have been reported previously (33). The
intron/exon junction also contains a recognition sequence for the
restriction enzyme XbaI (TCTAGA). The TFO used in these
studies, AE-07, was linked to psoralen and contained a patch of four
2'-AE substituted nucleotides, whereas the remainder of the molecule
contained 2'-OMe sugars (Fig. 1, a and b). We have described the synthesis and characterization of TFOs containing 2'-AE substitutions in previous publications (17, 18). The 2'-AE
residues are protonated at physiological pH. They reduce the charge
repulsion between the third strand and the duplex target, thus lowering
the requirement for Mg2+ (34, 35). They also establish a
stabilizing interaction with phosphates in the purine strand of the
duplex target (36). Psoralen-linked TFOs containing the appropriate
amount and distribution of this modification are active in the
Hprt assay (18).

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Fig. 1.
a, CHO Hprt target
sequence. The polypurine:polypyrimidine sequence is flanked by a
5'-TA step contained within an XbaI recognition site,
all adjacent to Exon 5 (small letters). The sequence of
AE-07 and the location of 2'-AE residues (all of the other sugars were
2'-OMe) are shown. b, the structure of the T and C
derivatives used in AE-07.
|
|
Pso-TFO Activity during the Cell Cycle--
AE-07 was introduced
by electroporation into quiescent CHO cells and into cells at different
times after release from quiescence (see "Materials and Methods")
(for fluorescence-activated cell sorter profiles of quiescent and mid S
phase cells, see Fig. 2a). The
psoralen was photoactivated, and the frequency of cells with inactivating mutations in the Hprt gene was determined via
conventional thioguanine selection. The mutation frequency was quite
low in the cells in G0/G1 but increased in
cells traversing G1 and appeared maximal in S phase cells.
To more precisely define the time of peak activity in S phase, the
cells were released from G0/G1 block and then
blocked again in early S phase by treatment with complete medium
containing mimosine (30). The cells were released from this block and,
as before, were treated at various times with AE-07/UVA followed by
determination of thioguanine-resistant colonies. The greatest activity
was seen 4-6 h after release during which time the cells were in
mid-to-late S phase. Results from selected time points are shown in
Fig. 2b. The mutation frequency of cells treated in mid-S
phase was 0.15-0.22%, whereas the level in quiescent cells was
0.02-0.03%.

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Fig. 2.
a, fluorescence-activated
cell sorter profile of G0/G1 (left
panel) and mid-S phase 4 h after release from mimosine block
(right panel) cells. b, frequency of
Hprt colonies after Pso-AE-07 treatment at
different times in the cell cycle (quiescent cells, G0;
4 h after release, G1; in mimosine block, early S;
4 h after release from mimosine, late S). The frequency with
untreated cells was ~0.001% regardless of cell cycle time.
c, frequency of mutant colonies following treatment with
free psoralen/UVA. Note the difference in scale on the y
axis in b and c. LS, late S;
CTL, control.
|
|
Psoralen Mutagenesis during the Cell Cycle--
Our previous work
(14) shows that the appearance of mutant cells required
photoactivation. Thus, the actual mutagen was the psoralen cross-link.
Consequently, it was of interest to question whether Hprt
mutagenesis by free psoralen would show cell cycle variability. We
treated cells at different phases of the cell cycle with psoralen (see
"Materials and Methods") and processed them as before.
Thioguanine-resistant colonies were recovered at frequencies a few fold
over the background (0.004-0.005 versus 0.001%),
consistent with similar experiments with psoralen by other groups (37,
38). However, in contrast to the previous experiment, the mutation
frequency was essentially unchanged across the cell cycle (Fig.
2c).
Electroporation Controls and Specificity--
Several control
experiments were performed. Cells at different times of the cycle were
treated with nonspecific TFOs or the specific TFO without
photoactivation, or they were mock-electroporated and UVA-treated. We
saw no increase in mutation frequency relative to cells that received
no treatment in any of these experiments. We also measured the
efficiency of electroporation in G0/G1 and S
phase cells using a green fluorescent protein reporter plasmid assay.
We found only a modest difference in efficiency in S phase (60-70%
positive cells) as compared with G0/G1 cells
(40-50%). The assay was repeated with fluorescent oligonucleotides.
Approximately, 50% G0/G1 cells showed nuclear
fluorescence versus 80% for those in the S phase, again too
small to explain the differences in Fig. 2b.
The Aprt gene has polypurine:polypyrimidine elements similar
but not identical to the Exon 5 target in Hprt. There are
straightforward selection protocols for identifying cells with
mutations in Aprt. We have used this assay to monitor the
specificity of the TFOs designed for the Hprt target. In
previous work (14), we found that although AE-07 treatment mutagenized
Hprt, it had no effect on Aprt. We repeated this
experiment with cells in mid-S phase but again found no increase over
background in the frequency of cells with mutations in the
Aprt gene (data not shown).
The results of these experiments suggested that the differences in
pso-TFO activity in quiescent and S phase cells were not artifacts of
electroporation efficiency differences or because of a loss of target
specificity (with an attendant increase in random mutagenesis)
in S phase. The indifference to cell cycle position of mutagenesis by
free psoralen indicated that there was a fundamental difference between
the two reagents. Two nonexclusive explanations for the variability of
the TFO-targeted mutagenesis appeared possible. The first explanation
that the frequency of TFO targeted cross-links was the same in
G0/G1 and S phases and that the kinds of
mutations or perhaps the efficiency of mutagenesis of the TFO
cross-links varied. Alternatively, the frequency of TFO-mediated
cross-link formation might be different in
G0/G1 and S phases.
Kinds and Frequency of Mutations Induced by pso-TFO--
The
published spectra of psoralen-induced mutations in Hprt are
dominated by base substitutions (37, 38). We have performed experiments
with mutation reporter plasmids carrying TFO-psoralen cross-links in
the Hprt target sequence embedded in the supF
reporter gene. These mutation profiles were also dominated by base
substitutions. Almost all were located at T of the 5'-TA cross-link
site (data not shown) (for schematic of the reporter gene, see Ref.
17). Targeted point mutations were also the major event in earlier studies (39, 40) with psoralen-linked TFOs and supF reporter genes. However, in our analysis of pso-TFO-targeted mutations at the
endogenous chromosomal Hprt target site, we found that ~90% thioguanine-resistant clones carried deletions in the target region extending into Exon 5 (14, 17) and no point mutations were
observed at the T of the 5'-TA cross-link site. This disparity could
result from marked differences in the processing of the pso-TFO
cross-link in the chromosomal target as compared with the shuttle
vector plasmid (and the free psoralen) or might simply reflect the
failure of the thioguanine resistance assay to report base
substitutions at this site.
We tested the latter possibility by isolating DNA from colonies chosen
at random following pso-TFO/UVA treatment (no selection was applied).
The PCR products of the I4E5 target region from these clones were
digested with XbaI whose recognition site is coincident with
the cross-link target site (Fig. 1a). We screened 800 colonies from S phase experiments and recovered 54 digestion-resistant clones (6.8%) (Fig. 3). The analysis was
performed on G0/G1 colonies, and 6 of 731 were
resistant (0.8%). Sequence analysis of resistant PCR fragments (43 S
phase and 4 G0/G1 phase) indicated that 41 of
43 were T
C at the T of the 5'-TA cross-link site (the others were
point mutations at adjacent bases within the XbaI site). Two
hundred colonies from mock-transfected/UVA-treated cells were also
analyzed. There were no XbaI-resistant clones. These results showed that the actual frequency of targeted mutations was much higher
(30-fold) than reported by the thioguanine selection. However, they
confirmed the disparity between the G0/G1 and S
phase mutation frequencies.

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Fig. 3.
XbaI digestion of PCR fragments of
the target region from non-selected colonies of cells treated with
AE-07/UVA in S phase. The arrow indicates the
undigested fragment.
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Pso-TFO-mediated Cross-linking in Synchronized Cells--
The
difference in targeted mutation frequency between quiescent and S phase
cells could be the result of cell cycle variability in mutagenesis
functions (41) or a difference in the efficiency of pso-TFO cross-link
formation. To test the latter possibility, we treated
G0/G1 and S phase cells with the pso-TFO and
UVA and then isolated total genomic DNA. We used the classical
denaturation resistance technique to measure the level of cross-links
in a restriction fragment containing the target sequence (42). The DNA
was digested with EcoRI, and the digests were denatured and electrophoresed on neutral agarose gels. The gels were blotted onto a
nylon filter and hybridized with a probe specific for the 2-kb fragment
containing the target sequence. In initial control experiments,
purified genomic DNA was incubated with the pso-TFO and was UVA-treated
in vitro, restricted, and then either run separately or
mixed with an equal amount of untreated DNA. The hybridization pattern
showed clear resolution between the cross-linked and non-cross-linked
samples and run separately or in mixture (Fig.
4a). The hybridization signal
of the cross-linked fragment was ~50% of the non-cross-linked
control, reflecting the reduced efficiency of hybridization and/or the
reduced retention on the filter of cross-linked DNA. The analysis of
the DNA from the treated cells showed a greater extent of cross-linking
in S phase than in G0/G1. Quantitation by
PhosphorImager indicated that ~19 ± 2% S phase DNA was
cross-linked (seven experiments), whereas the G0 level was
4 ± 2%. To control for nonspecific cross-linking, the blots were
stripped and rehybridized with a probe to a 3-kb fragment of the
Dhfr gene (Fig. 4b). There were no
denaturation-resistant fragments in any of the samples, suggesting that
the resistance was specific to the fragment containing the target
sequence.

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Fig. 4.
a, denaturation resistance assay of
targeted cross-links. Genomic DNA treated in vitro or
in vivo with AE-07, was digested with EcoRI,
which releases a 2-kb fragment containing the target site. Samples with
the exception of lane 4 were denatured and electrophoresed,
blotted, and hybridized with a probe to the 2-kb EcoRI
fragment. Lane 1, genomic DNA cross-linked in
vitro by AE-07; lane 2, non-cross-linked DNA;
lane 3, mixture of equal amounts of lanes 1 and
2; lane 4, non-cross-linked, non-denatured DNA.
Denatured DNA from untreated control cells (lane C) or
AE-07-treated G0/G1 or S phase cells (two
independent experiments). The arrowhead marks the position
of the denatured fragment, and the arrow marks the
non-denatured or denaturation-resistant fragment. b,
hybridization with a probe to a 3-kb (arrow) fragment of the
DHFR gene. Samples were not denatured
(CND) or denatured from control (C) or
AE-07-treated G0/G1 or S phase cells.
c, blot of EcoRI and XbaI digestion of
samples from AE-07-treated cells. Control DNA digested with
EcoRI (CR) or EcoRI and
XbaI (SRX or CRX). DNA
from AE-07/UVA G0/G1 or S phase cells digested
with EcoRI and XbaI. The arrow marks
the position of the XbaI-resistant fragment.
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|
The difference in targeted TFO-psoralen adducts in cells in S phase or
G0/G1 was also shown by restriction digestion
protection. DNA from treated cells was digested with EcoRI
and XbaI followed by blotting and hybridization. The level
of the XbaI-resistant fragment was severalfold higher in the
S phase (25-30% protection) sample than G0/G1
(4-5%) (Fig. 4c). The greater signal from this assay may
reflect the inhibition of the restriction enzyme by both psoralen
mono-adducts and cross-links, whereas only the cross-links are reported
by the denaturation resistance assay.
 |
DISCUSSION |
The pso-TFOs (43) are two component reagents with the extent of
cross-linking (and the mutational consequences thereof) dependent upon
and serving as a measure of the bound TFO at the time of
photoactivation. The data presented here and in our previous publications (17, 18) show that both the chemistry of the oligonucleotide and the biology of the cell are important determinants of TFO binding at a chromosomal target.
The biochemical measurement of the frequency of targeted cross-links
combined with the frequency and kinds of mutations affords an initial
insight into the relative importance of the several pathways available
for processing cross-links. Approximately, 20-30% of the target
sequences were cross-linked when the cells were treated in S phase.
However, the overall mutation frequency was ~7%. This indicates that
as many as 75% of the cross-links were resolved without mutagenesis.
>95% of the mutations were single base substitutions, whereas the
deletions accounted for the remainder. Mutagenesis and repair of
psoralen cross-links have been examined by many groups, and multiple
pathways are involved (44-48). Current models involve the generation
of single strand gaps at the cross-link site, perhaps requiring the
activity of the XPF·ERCC1 (XPF, the gene mutated in
Xeroderma pigmentosum, complementation group F; ERCC1,
excision repair cross complementing gene 1) complex (49).
Recombinational repair of the gap using another intact copy of the
target region as a donor would yield an unaltered version of the
original target. Alternatively, the gap could serve as a template for
synthesis by one or another of the damage bypass polymerases (50). This
synthesis could have mutagenic consequences or might be error-free.
Presumably, this would be the source of the point mutations. A second
cycle of excision repair and synthesis would eventually produce either a mutant or wild type target sequence. Our base substitution data are
in good agreement with a previous study (51) of pso-TFO mutagenesis of plasmids in yeast that elucidated mutagenesis from the
furan or pyrone side of the psoralen cross-link (51, 52). This work
showed mutation patterns consistent with preferential excision of the
duplex on the transcribed strand and also on the furan side of the
cross-link. When both conditions applied as would be the case with the
I4E5 target, the mutations were generated at the T at the pyrone
side of the cross-link.
The majority of thioguanine-resistant clones contains deletions in
which the psoralen target site and flanking sequence are lost
(occasionally, we recover mutations at the A of the TA cross-link site). The deletions extend at least through the splice acceptor site,
the minimum required to inactivate the gene. Our earlier sequence
analysis of the deletions suggested that most of these were derived
from double strand breaks (14, 17). Deletions could be caused by
successive cycles of incisions by excision repair enzymes or as a
consequence of collisions with replication forks (53). Whatever the
molecular pathways for the deletions, they are exercised much less
frequently than those producing point mutations.
The recombinational repair pathway would not be operable in quiescent
cells but would be available during the mid-S phase of our experiments
as Hprt replicates early in S phase (54, 55). The similar
ratios of base substitution and deletion mutations (albeit at different
frequencies) from G0/G1 and S phase cells may
indicate that the same mutation pathways operate in both conditions. Alternatively, it is also possible that the mutant cells recovered from
the G0/G1 cells were actually derived from a
small number of S phase cells that escaped quiescence.
Why the greater frequency of targeting in S phase? There would seem to
be at least three possible explanations. One is that the
G0/G1 cells are inefficiently electroporated
relative to S phase cells. Our analysis of the electroporation
efficiencies of the green fluorescent protein plasmid and the
fluorescent oligonucleotides does not appear to support this
contention. It is true that the physical presence of the fluorescent
oligonucleotide in the nuclei of the G0/G1
cells is not evidence of a functional presence. However, the green
fluorescent protein plasmid results clearly reflect a functional,
nuclear presence, and our experience has been that plasmid and
oligonucleotide electroporation efficiencies are well correlated. A
second possibility is that the stability of triplexes once formed is
much greater in S phase cells than G0/G1 cells. We have shown that triplexes are less stable in cells than in "physiological buffer" in vitro, perhaps because of
cellular enzymes that can disrupt triplexes (56). These include
helicases and the translocases found in chromatin-remodeling complexes
(57-59). However, this suggestion is somewhat counterintuitive since
we generally associate chromatin remodeling with gene activation and
Hprt gene expression is quite low in quiescent cells
(60).
A third possibility is that the levels of TFO binding reflect the
accessibility of the target sequence. Triplex formation by 15-20
nucleotide TFOs on nucleosomal sequences is impeded by the requirement
of the third strand to occupy the major groove, 8-10 base pairs of
which would be exposed with the remainder turned against the histone
core complex. In vitro studies indicate that nucleosomal
sequences associated with the nucleosomal core are inaccessible to
TFOs, whereas those at the ends or in internucleosomal linker regions
are more available (20-22, 61). Although direct experiments have not
been performed, one implication is that nucleosomal DNA whose
association with the histone core has been relaxed by ATP-dependent remodeling (62-64) or certain histone
modifications (65) would be more open for triplex formation. The
Hprt gene is a member of a group of constitutively expressed
housekeeping genes (including DHFR) whose expression is
quite low in quiescent cells (60, 66). Transcript levels increase
markedly upon growth stimulation and, in the case of DHFR,
are dependent on E2F transcription factors (67). This family
also includes repressors of E2F-dependent promoters,
which are involved in silencing genes in quiescent cells via mechanisms
that include alterations in histone modification and chromatin
structure (68, 69). Assuming that these alterations extend to
nucleosomes downstream of the promoter region, it may be that TFO
bioactivity reflects the chromatin structural dynamics of the
Hprt target.
The results presented here have practical significance. TFOs have been
of interest for many years because of potential applications as
gene-targeting reagents for manipulation of genomic sequences. The
Hprt gene target has been instrumental in the development of
bioactive TFOs because of the opportunity for facile and quantitative selection of cells with targeted events. However, most genes of interest do not have a selection option available for identification and analysis of cells with the desired end point. Thus, it will be
important to develop protocols that permit recovery of clones of
interest with a minimum of effort. The data presented here suggest that
successful procedures will require effective oligonucleotide chemistry
combined with manipulation of the cell biology. This will be an
important consideration for gene targeting in hematopoietic stem cells,
which are largely quiescent (70).
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M211837200
The abbreviations used are:
TFO, triple-helix
forming oligonucleotides;
2'-OME, 2'-O-methyl;
pso-TFO, TFOs
linked to the DNA cross-linker psoralen;
2'-AE, 2'-O-aminoethyl;
CHO, Chinese hamster ovary;
UVA, ultraviolet A;
kb, kilobase.
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