Pharma Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
Received 18 November 1997/Returned for modification 12 January
1998/Accepted 21 May 1998
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INTRODUCTION |
Few homologous recombination methods
have thus far been described and applied to generate subtle
modifications in the mammalian genome. Two procedures have common
features, namely, modification of the targeting locus in a first step
and then allowing for selection for the modification in a second step.
The "targeting-in-out" procedure requiring two subsequent
replacements at the locus of interest had been described for
introduction of subtle mutations into the 
2-Na,K-ATPase
(2) and Col1a-1 (22) genes of mouse embryonic
stem (ES) cells. Another procedure, termed "hit and run," which
makes use of an intermediate genomic duplication was described for
subtle changes at the Hox-2.6 locus (11) and for a directly
selectable insertion into the hypoxanthine phosphoribosyltransferase gene of mouse ES cells (21). Both procedures depend entirely on two homologous recombination steps. Another system, the
Cre-loxP system (reviewed by Marth [14]),
can also be used for subtle gene targeting, yet after introduction of
loxP sequences into the desired genomic locus by homologous
recombination, Cre-recombinase then induces a site-specific
recombination between the loxP sequences.
In the present study, pathogenic 
-amyloid precursor protein
(-APP) mutations known to segregate with a familial form of early-onset Alzheimer's disease (AD) in Sweden (SFAD) (15)
or with hereditary cerebral hemorrhage with amyloidosis in a Dutch family (HCHWA-D) (12) were introduced into mouse ES cells.
These two mutations are located on exons 16 and 17 in the -APP gene like all other pathogenic -APP mutations known so far (for an overview, see the report of Schellenberg [17]). Gene
targeting was approached in the present study by homologous
recombination between the cellular -APP gene and mutant targeting
vectors by using a targeting-in-out and a hit-and-run procedure. The
efficiencies and characteristics of both homologous recombination steps
of both methods were evaluated and compared. The targeted mutated ES
cells may be used to establish mice with a mutant -APP gene under
the control of endogenous genomic regulatory sequences without background wild-type gene expression.
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MATERIALS AND METHODS |
Isolation and characterization of -APP mouse genomic
clones.
All molecular biological methods not explained in detail
here were performed by standard techniques (6, 16). Lambda
phage clones containing exons 16 and 17 of the -APP gene were
obtained by screening a C57BL/6 mouse brain genomic library in
EMBL3a, a kind gift from Wolfgang Wille (Universität
Köln, Cologne, Germany). Phage plaques were transferred in
duplicate to nylon membranes (GeneScreen Plus; DuPont NEN), and
denatured DNA was tested with two different probes, i.e., one on each
filter. Probe 1, an oligonucleotide complementary to the 3' end of
human -APP exon 18 (5'-GGCGGGGGTCTAGTTCTGCATCTGCTCAAAGAACTTGTAGGTTGG-3') was
end labeled with [-32P]dATP by using terminal
deoxynucleotidyl transferase and hybridized to membrane-bound DNA for
16 to 30 h at 60°C in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1× Denhardt's solution with 10 µg of carrier DNA
per ml. Membranes were washed with 0.5× SSC-0.1% sodium dodecyl
sulfate (SDS) at 52°C prior to autoradiography. As a second probe, a
241-bp HincII rat cDNA fragment covering exons 16 and 17 was
random prime labeled with [-32P]dCTP by using the
T7QuickPrime kit (Pharmacia). Hybridization for the second
probe was done in 4.8× SSC-20 mM Tris-HCl (pH 7.4 at 20°C)-45%
formamide-8% dextran sulfate-1× Denhardt's solution-0.1% SDS for
16 to 30 h at 42°C. Membranes exposed to this probe were washed
with 0.2× SSC-0.1% SDS at 58°C prior to autoradiography.
Double-positive phage plaques were enriched and purified, and inserts
were subcloned into plasmid pBluescriptII SK
(Stratagene). These
clones were further analyzed by restriction mapping and double-strand
sequencing by using the dideoxy chain termination method with Sequenase
(U.S. Biochemicals) and the Bst sequencing kit (Bio-Rad).
Construction of targeting vectors.
Site-directed mutagenesis
was performed with the Transformer kit (Clontech). Desired mutations
within exons 16 and 17 of -APP were introduced into mouse genomic
subclones in pBluescriptII by using the mutator oligonucleotide Mut-1
(5'-GATCTCGGAAGTGAATCTAGACGCGGAGTTCGGACATGATTCAG-3') for the SFAD mutation on exon 16 or Mut-2
(5'-GAAGGTGTTCTTTGCGCAGGACGTCGGATCGAACAAAGGCGC-3') for the HCHWA-D mutation on exon 17 (underlined letters indicate nucleotide exchanges). Simultaneously, the unique NotI
restriction site in the polylinker region was deleted for selection
with the oligonucleotide Sel-1
(5'-CCGCGGTGGCAGCTGCTCTAGAAC-3').
Targeting vectors were constructed by using an 11-kb genomic
-APP fragment containing modified exons 16 and 17 starting 1.5 kb
upstream of exon 16 and ending at the 5' splice site of exon 18 (Fig.
1). Genes for neomycin resistance (Neo)
and thymidine kinase from herpes simplex virus (TK) (3) were
joined to form a selection cassette and attached to the genomic -APP
fragment. The Neo gene of this cassette, derived from transposon
Tn5 (4), is under control of the mouse
phosphoglycerate kinase promoter and polyadenylation signal
(1). It was originally cloned by Michael McBurney (Ottawa, Ontario, Canada) and provided by Colin L. Stewart (Nutley, N.J.). The
TK gene was excised from the vector plC19R/MC1-TK (constructed by Suzi
Mansour, Salt Lake City, Utah) (13). For the replacement vector pBL-RV(in), the selection cassette was cloned into the HindIII site of the genomic -APP fragment (1.0 kb
downstream of exon 17) in pBluescriptII (Fig. 1A). No selection genes
were put into the replacement vector pBL-RV(out) designed for a second transfection of ES cells that already contained TK and Neo after a
preceding transformation (Fig. 1A). The insertion vector
pGEM-IV(hit-run) was constructed by recircularization of the
SalI-excised genomic -APP fragment followed by
linearization with KpnI (2.0 kb downstream of exon 17) and
subcloning it into pGEM7 ZF (Promega). The selection cassette was
then inserted at the reopened SalI site. Prior to transfection, all targeting vectors were linearized and excised from
pBluescriptII or pGEM7 with SalI or KpnI,
respectively.
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FIG. 1.
Approaches for introduction of subtle mutations into ES
cells by homologous recombination. (A) The targeting-in-out procedure.
Boxes with numbers indicate the positions of exons. Those exons
carrying the SFAD (16S) or HCHWA-D (17H)
mutation are highlighted, and mutated -APP alleles are labeled by
the same superscript letters. Boxes with text and arrowheads indicate
the positions and orientations of selection genes. The locations of
crossovers during homologous recombination are indicated by crosses.
After the first homologous recombination (targeting IN), ES cells
having replaced the targeted locus by the replacement vector RV-S(in)
are enriched by selection for neomycin resistance (Neo) with G418.
Isolated targeted-in ES cells are subsequently targeted OUT again by
homologous recombination with RV-H(out) and selection with FIAU against
the viral TK. Four possible end arrangements are shown, three of which
contain mutations but lack the selection cassette. (B) The hit-and-run
procedure. Selection of hit clones with a partial genomic duplication
due to targeted integration of the vector IV-H(hit-run) by homologous
recombination is performed as in the targeting-in step. After the run,
ES cells that had undergone spontaneous intrachromosomal recombination
resulting in loss of the duplication and selection cassette are
counterselected with FIAU against TK. This process ends up either in
reversion to wild-type (wt) or in expected targeted mutated ES cells
without a selection cassette. (C) Primers and probes. Probes 3 to 6 (P3
to P6) for genomic Southern blot analysis are indicated by dark solid
lines, and the genomic fragment used for construction of targeting
vectors is shown as a dotted line. The binding sites and 5'-to-3'
orientation of primers used for PCR are shown by arrowheads. The
positions of recognition sites for restriction endonucleases are
indicated by single letters: A, AatII; B, BamHI;
C, ClaI; H, HindIII; K, KpnI; N,
NotI; S, SalI; X, XbaI.
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Cell culture.
E14 ES cells derived from mouse strain 129/Sv
with a karyotype of 40XY were provided by Horst Blüthmann (Basel,
Switzerland). To prevent totipotent ES cells from differentiating, they
were cultivated permanently on a feeder layer of irradiated primary mouse embryonic fibroblasts (PMEFs) transgenic for the Neo gene. PMEFs
were prepared from 13-day-old ICR-M-TKneo2 embryos (19) and
kept for a maximum of seven passages in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 2 mM
L-glutamine, 1× minimal essential medium nonessential
amino acids (Gibco), 100 U of penicillin-streptomycin (Gibco) per ml, and 100 µM -mercaptoethanol at 37°C under 10% CO2.
Prior to serving as feeders for ES cells, PMEFs were allowed to build a
nearly confluent monolayer and were inactivated by irradiation with
3,000 rads by using a cesium source. E14 cells were routinely passaged every 2 to 3 days and cultured in the same medium as that used for
PMEFs alone, but supplemented with 103 U of leukemia
inhibitory factor (ESGRO; Gibco) per ml and with 15% fetal calf
serum.
Transfection and selection of ES cells.
For transfection,
2 × 107 ES cells were resuspended in 800 µl of
ice-cold phosphate-buffered saline with 20 to 25 µg of linearized targeting vector and pulsed in cuvettes with a 0.4-cm electrode gap at
280 V and 500 µF with Genepulser electroporation equipment (Bio-Rad).
After electroporation, cells were taken up in culture medium without
selective drugs and seeded at a density of 2 × 106 to
4 × 106 treated cells per 6-cm-diameter culture dish
on an irradiated feeder. For positive selection, i.e., for cells
expressing the Neo gene, culture medium was replaced 24 h after
electroporation with selection medium containing in addition 175 µg
of G418 (Geneticin; Gibco) per ml. E14 cells were kept for 9 days under
G418 selection conditions with fresh medium supplied every day before
single ES colonies were picked by using glass capillary mouth pipettes. Counterselection for ES cells having lost the viral TK gene previously cointroduced by positive selection was performed by supplementation of
medium with 0.2 µM fialuridine (FIAU; Oclassen) after a period of 1 to 5 days in nonselective medium. This negative selection was done for
7 days either directly for cells targeted with insertion vectors or
when using replacement vectors after electroporation with a second
replacement vector lacking the TK gene. Medium was also renewed every
day, and single ES colonies were picked as described above.
Analysis of targeted ES cells by PCR.
DNA was extracted from
1 × 103 to 2 × 103 cells (ES cells
cultured on PMEFs) in 10 µl of distilled water by using 100 µl of InstaGene purification matrix (Bio-Rad) as described in the
manufacturer's instructions. Lysate aliquots of 20 µl were used as a
template for each 50-µl PCR mixture corresponding to 200 to 400 cell
equivalents. All PCRs were performed by using the Expand Long Template
PCR system with buffers 1 and 2 (Boehringer Mannheim) under modified conditions. Reactions were performed in thin-walled 500-µl tubes by
using a TRIO Thermoblock Cycler (Biometra), and the enzyme mix was
added after the initial DNA denaturation at 92°C for 2 min. Fifteen
cycles of 92°C for 10 s, 65°C for 30 s, and 68°C for 2 to 6 min (depending on length of the expected fragment) were followed
by another 25 cycles, with elongation times increasing for 20 s
each round. All primers used for PCR are listed in Table 1, and binding sites are indicated in
Fig. 1C. Mutation-specific fragments were selectively amplified and
visualized directly by agarose gel electrophoresis of the PCR mixtures.
Genomic Southern blot analysis.
After extraction of genomic
DNA from cells (ES cells cultured on PMEFs) in accordance with standard
procedures, samples containing 5 µg of DNA were digested for 5 to
10 h with different combinations of restriction enzymes as
described in the manufacturer's instructions (Boehringer Mannheim).
After agarose gel electrophoresis, DNA was denatured and blotted by
capillary transfer to nitrocellulose membranes (BA85, 0.45-µm pore
size; Schleicher & Schuell). For hybridization, four genomic mouse
-APP restriction fragments specific for intron 15 (probe 3), intron
16 (probe 4), intron 17 (probe 5), and exon 18 (probe 6) were used
(Fig. 1C). These probes were random prime radiolabeled and hybridized
to the immobilized DNA as described above for probe 2. Autoradiography
was performed after washing the membranes with 0.2× SSC-0.1% SDS at
50 to 70°C depending on the background radioactivity.
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RESULTS |
Genomic structure of the 3' end of the mouse -APP gene.
A
genomic clone which contains an insert of 12.5 kb starting 1.5 kb
upstream of exon 16 and ending 200 bp downstream of the 3' noncoding
region of exon 18 was isolated. Sequencing of exons 16 to 18 showed
100% nucleotide identity to the previously reported cDNA sequence from
mouse strain BALB/c, including the large 3' noncoding region of exon 18 (9). The exon-intron boundaries of exons 15 to 18 were found
at the same positions as in humans (23) and rabbits
(5). The position of exon 15, which was absent on the
characterized genomic clone, was determined to be 5.6 kb upstream of
exon 16 (Fig. 1) by PCR by using primers 15F and 16R-wt (Table 1).
Introduction of mutations into ES cells by targeting in-out.
For the first step of this approach (Fig. 1A), ES cells were
transfected with the replacement vector RV-S(in), an 11-kb genomic -APP fragment carrying the SFAD mutation and a selection cassette with Neo and TK genes. Forty to 60% of the 2 × 107
ES cells electroporated with 20 µg of targeting vector at 280 V and
500 µF survived this procedure. Selection for cells that had
integrated the replacement vector was started after 24 h by addition of the antibiotic G418. While mock-transfected ES cells were
eliminated completely within 5 days of selection, G418-resistant (G418r) ES cell colonies became visible after 4 to 5 days.
Counting of G418r colonies after a total selection time of
9 days showed a frequency of 1.4 × 10
4 to 2.0 × 104 per cell surviving electroporation for integration
of the Neo gene located on the replacement vector into the genome.
Single colonies were picked, expanded, and analyzed for homologous
recombination events by PCR by using primers 15F and 16R-S (Table 1).
This detected specifically the SFAD mutation at the expected position
on exon 16, i.e., 5.7 kb downstream of exon 15 (Fig. 1). By agarose gel
electrophoresis, 17 to 21% of the G418r cells were found
to be PCR positive (Fig. 2A, left panel).
After PCR with primers 16F-S and 17R (Table 1), the expected
mutant-specific band at 2.8 kb was obtained from all clones previously
identified; this band would, however, also be obtained from random
vector integrations (Fig. 2A, right panel). For verification of these results, Southern analysis of XbaI-restricted genomic DNA
from isolated ES cell clones was performed by using intron 15-specific probe 3, which is unable to bind directly to the targeting vector (Fig.
1C). Thus, the correctly positioned SFAD mutation would be detected by
probe 3 hybridizing to an additional mutation-specific restriction
fragment. This fragment is shorter by 1.8 kb than the wild-type
fragment because of the new XbaI site on exon 16 of the
targeted allele. All clones identified in the first PCR screen were
verified by this method (Fig. 2B, left panel), excluding the
possibility of false-positive signals caused by random integrations of
the targeting vector and primer extension during PCR (10). However, the intensities of bands representing wild-type and mutant alleles are not equal. The genomic DNA was extracted from cocultures of
ES cells with inactivated feeder cells (mouse embryonic fibroblasts), and the upper wild-type band is thus generally expected to be more
intense; it derives from the wild-type allele of ES cells and from both
alleles of feeder cells. Indeed, Southern blot analysis of tail biopsy
samples from chimeric mice generated by using these targeted-in ES cell
clones showed mainly equal ratios of wild-type to mutant bands
(unpublished data). The possibility of additional randomly integrated
targeting vectors was also tested by Southern analysis.
HindIII-restricted genomic DNA from targeted-in ES cell clones was hybridized with the intron 16-specific probe 4 (Fig. 1),
which detected the HindIII site 1.8 kb upstream of exon
16 that is not present on the targeting vector that begins just 300 bp
upstream of this restriction site. A single band at 5.6 kb, corresponding to the wild type and the SFAD mutant, was detected. Additional bands of different lengths, as consequences of random integrations or vector repeats, were lacking in all clones tested (Fig.
2B, right panel). Southern blot analysis was also used to verify the
correct position of the selection cassette by using probe 6, which is
specific for exon 18, on ClaI-restricted DNA (Fig. 1). As
expected, an additional 7.2-kb band was found for the mutant allele
while the wild-type signal was larger than 15 kb (data not shown).
Thus, with the vector RV-S(in), the frequency for targeted homologous
recombination, coupled with mutagenesis of exon 16 and integration of
the selection cassette, was 2.9 × 105 to 3.3 × 105 per viable cell after electroporation under the
conditions used.
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FIG. 2.
Characterization of targeted-in ES cells (clones 2, 11, 15, 22, 41, and 48). (A) Identification of positive ES cell clones by
screening with SFAD-specific PCR by using primer pairs 15F-16R-S and
16F-S-17R (left and right panels, respectively). PCR products were
analyzed directly by electrophoresis of 20-µl aliquots of the
reaction mix on 1% agarose gels. The mutant-specific bands at 5.7 and
2.8 kb indicate the distance of the SFAD mutation, located on exon 16, from the neighboring exons 15 and 17. (B) Verification of positive ES
cell clones by Southern blot analysis. Genomic DNA isolated from
positive ES cell clones (cultured on PMEFs) was digested with either
XbaI or HindIII, separated by electrophoresis
on 1% agarose gels, and hybridized with intron 15-specific probe 3 or
with intron 16-specific probe 4 after transfer to a nitrocellulose
membrane. An additional 8.2-kb band detected with probe 3 on
XbaI-restricted DNA indicated the targeted mutation by
homologous recombination with RV-S(in) in PCR-positive cells (left
panel). Because the preparations also contained DNA from feeder cells,
the upper wild-type band appeared generally more intense than the
8.2-kb mutant band. For exclusion of additional random integrations of
targeting vectors, HindIII-restricted DNA was tested
with probe 4. Except for the 5.6-kb band for the wild type (wt) and
SFAD, no additional bands indicative of random integrations of RV-S(in)
were found (right panel).
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Clones of "hit" ES cells with the targeted mutation were further
expanded and kept under G418 selection to prevent spontaneous reversion
to wild type. For targeting out, mutated ES cells were transfected with
the replacement vector RV-H(out), a simple genomic fragment
modified by the HCHWA-D mutation on exon 17. The purpose of this second
transfection was to introduce an additional mutation (HCHWA-D) and to
replace the nearby selection cassette with the original intron sequence
(Fig. 1A). For selection against the viral TK gene located on the
selection cassette, the culture medium of transfected ES cells was
supplemented with 0.2 µM FIAU 3 to 4 days after electroporation.
While growth of wild-type ES cells was not affected by this treatment,
TK-positive clones were killed within 2 to 4 days of selection. After 5 days, single FIAU-resistant (FIAUr) colonies became visible
and were picked after 7 days of selection. With 0.8 × 103 to 4.4 × 103 resistant colonies
per viable cell after electroporation, FIAUr colonies
appeared up to 25 times more frequently after the targeting-out step
than did G418r colonies after the targeting-in step.
Nevertheless, the frequency still indicates a stringent selection for
TK-negative cells. If the detected stronger intensities of the 10-kb
wild-type bands for targeted-in clones in Fig. 2 had not originated
from inactivated feeder cells but from a large fraction of wild-type ES
cells in targeted-in clones, the observed selection would not have
occurred. No correlation between a higher intensity of the wild-type
band and the rate of FIAUr clones after targeting out was
found. All FIAUr clones subjected to another round of G418
selection were killed, and no surviving subclones grew, verifying the
loss of the complete selection cassette by the targeting-out step.
After the selection, FIAUr clones were screened by PCR with
primers 17F-H and 17+R (Table 1) for integration of RV-H(out) containing the HCHWA-D mutation (Fig. 1A). Random integrations would be
detected as well, but only homologous recombination with the correct
locus and replacement of the targeted-in TK gene should make
targeted-out cells FIAUr, for which they were preselected.
In testing 384 FIAUr ES cell clones, derived from 12 different targeted-in clones, not a single PCR-positive HCHWA-D clone
was detected. PCR conditions were sensitive enough because addition of
RV-H(out) directly to negative cell lysates prior to PCR gave strong
2.1-kb signals, even for a copy number 10 times smaller than expected
for a targeted clone (data not shown). In addition, equally strong PCR
signals were obtained from cell lysates for wild-type control with
primers 17F-wt and 17+R (Table 1; Fig. 1), indicating sufficient
quality of the DNA templates (data not shown). However, after the
targeting out, FIAUr clones could still have retained the
SFAD mutation and lost only the selection cassette. This possibility
was tested by SFAD-specific PCR with primers 15F and 16R-S as in the
targeting-in step. Again no positive clones were found, suggesting that
all clones have reverted to wild type. Therefore, if all intermediate
clones obtained by the targeting-in step were equally capable to
undergo targeting out, the frequency of the second recombination step
was less than 1.1 × 105 per viable cell after
electroporation. Moreover, identical frequencies of FIAUr
colonies were observed for ES cell clones targeted out with RV-H(out) and for mock-transfected clones, indicating a process independent of a
homologous recombination with RV-H(out).
Targeting mutations by use of the hit-and-run technique.
The
hit-and-run approach represents a different strategy for introduction
of subtle mutations. Although this procedure requires two sequential
recombinations as does the targeting in-out method, only one
transfection, at the beginning, needs to be done (Fig. 1B). For this
hit step, the ES cells were electroporated with the targeting vector
IV-H(hit-run) as described above. The insertion vector contained the
same 11-kb genomic DNA fragment as the replacement vectors, with the
HCHWA-D mutation on exon 17. However, the ends of the fragment were
connected by the selection cassette and the targeting vector was
linearized within the genomic region by introduction of a double-strand
break 2.0 kb upstream of exon 17 (Fig. 1B). Selection with G418 for 9 days resulted in 1.2 × 104 to 4.5 × 104 resistant ES colonies per viable cell after
electroporation, which is similar to the frequency obtained with the
corresponding replacement vector RV-S(in) (1.4 × 104 to 2.0 × 104).
Single G418r clones were screened for the correctly
positioned HCHWA-D mutation on exon 17 by PCR by using primers 17F-H
and 17+R (Fig. 1). Both primers could also bind to the targeting vector but in the wrong orientation for direct PCR. Of the tested cells, 23 to
25% gave a correct 2.1-kb PCR fragment (Fig.
3A). Misleading signals could possibly
arise by primer extension (10) on randomly integrated
insertion vectors or by amplification from randomly integrated
head-to-tail tandem repeats of IV-H(hit-run) as frequently reported in
transgenic animals with multiple integrations of the transgene
(20). This was evaluated by Southern analysis with intron
17-specific probe 5 (Fig. 1) on XbaI-restricted DNA from potential hit clones. Only a single strong band at 3.2 kb was observed
for all clones tested (Fig. 4C). The same
pattern (single band at 6.8 kb) was detected by probe 5 hybridization
to DNA cut with BamHI (data not shown). Since no additional
fragments were found, random integrations can be excluded. However, the
intensities of the 3.2- and 6.8-kb signals were clearly elevated in
some clones relative to others, indicating a higher copy number of
-APP intron 17. Indeed, the presence of vector repeats, integrated
site specifically at the targeted locus, was confirmed in these clones
by Southern analysis of NotI-restricted DNA with intron
16-specific probe 4 (Fig. 4B). Only from the clones with tandem repeats
of IV-H(hit-run) could an intron 16-containing 11-kb genomic fragment
be excised between two neighboring selection cassettes, since those
were flanked by restriction sites for the rare-cutting enzyme (Fig. 1).
Finally, the exact position of the HCHWA-D mutation on the hit clones
was determined. The mutation on exon 17 could be located on both sites
of the partial genomic duplication (Fig. 1B) or even at several
positions in those clones with a targeted integrated tandem
repeat of IV-H(hit-run). A PCR with primers 15F and 17R-H was used to
detect the HCHWA-D mutation at the 5' site of the duplication, and
another reaction with primers NeoF and 17R-H detected the mutation
downstream of the selection cassette (Fig. 1B). The signal for
either the 5'-located mutation (8.5 kb) or the 3'-located mutation (5.3 kb) or both were found in all clones tested (Fig. 4A). Thus, all
12 clones analyzed were hits and contained targeted integrations of
IV-H(hit-run). Five of them contained targeted integrated tandem
repeats with the HCHWA-D mutation at different positions. The
others had integrated a single copy of the targeting vector, i.e., six
clones with the mutation only at the 3' site of the duplication and one
with HCHWA-D only at the 5' site. Considering all hit clones (including
those with insertion vector repeats), the frequency for targeted
integration of IV-H(hit-run) together with the HCHWA-D mutation by
homologous recombination was 3.0 × 105 to 5.0 × 105 per viable cell after electroporation. This
indicates similar efficiencies for the first steps of both targeting
approaches.
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FIG. 3.
Screening for hit-and-run ES cells with the HCHWA-D
mutation. (A) Identification of potential hit ES cell clones by
screening with an HCHWA-D-specific PCR by using primers 17F-H and 17+R.
PCR products were analyzed directly by electrophoresis of 20-µl
aliquots of the reaction mix on 1% agarose gels. The mutant-specific
band at 2.1 kb indicates the distance between the HCHWA-D mutation on
exon 17 and the binding site of primer 17+R (17H...)
located on the other side of the double-strand break in the 11-kb
genomic fragment of the insertion vector IV-H(hit-run). (B) Detection
of ES cell clones retaining the HCHWA-D mutation in their genome after
the run of the hit clone 110. The same PCR conditions and analysis used
for detection of hit clones were used for screening of run clones. wt,
wild type. Numbers at tops of lanes are clone designations.
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FIG. 4.
Characterization of potential hit ES cells. (A) Analysis
of the targeted locus of hit clones. The position of the HCHWA-D
mutation on the partial genomic duplication of hit clones was
investigated by two mutation-specific PCRs by using primer pairs
15F-17R-H for detection of the 5' location and NeoF-17R-H for the 3'
location. A combination of 15-µl aliquots from both reaction mixtures
was analyzed directly by electrophoresis on 0.6% agarose gels. The
band at 8.5 kb indicates the site-specific 5' position of the HCHWA-D
mutation, and the 5.3-kb band indicates the mutation when located 3' of
the selection cassette. (B) Detection of possible repetitive
integrations of the targeting vector by Southern blot analysis. Genomic
DNA isolated from positive ES cell clones was digested with
NotI, separated by electrophoresis on 0.6% agarose gels,
and hybridized with intron 16-specific probe 4 after transfer to a
nitrocellulose membrane. The appearance of a band at 11 kb indicated
the presence of head-to-tail tandem repeats of IV-H(hit-run) in some
clones. Because of incomplete NotI restriction, a 14.9-kb
band was detected together with the expected 11-kb band, showing
elongated fragments, i.e., products with an attached selection
cassette. (C) Exclusion of additional random integrations of targeting
vectors. XbaI-restricted DNA was run on 0.9% agarose gels
and tested with intron 17-specific probe 5. Except for the 3.2-kb band
for the wild type and for alleles with site-specific integrations of
IV-H(hit-run), no additional bands indicative of random integrations
were detected. However, because of the higher copy number, stronger
3.2-kb signals were obtained from those clones with specifically
integrated vector repeats. wt, wild type. Numbers at tops of lanes are
clone designations.
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Hit ES cell clones were expanded and kept under G418 selection before
the second homologous recombination step, the run, was initiated. Hit
clones were replated, and withdrawal of G418 allowed survival of cells
losing their selection cassette with the Neo gene. After 1 to 5 days in
nonselective medium, the counterselection against ES cells still
retaining the selection cassette with the TK gene was started by the
addition of FIAU to the culture medium and was maintained for 7 days.
As expected, the longer that ES cells were kept in nonselective medium,
the more FIAUr colonies were obtained. A constantly low
number of clones survived the FIAU selection after a selection-free
period of 1 to 3 days (1.8 × 104 to 3.9 × 104 per plated cell). After 4 days without selection, the
number of FIAUr clones per plated cell increased
drastically (5.5 × 102), and after 5 days, it was
~1.2 × 101. This indicates the presence of
residual TK activity that kills cells up to 3 days after loss of the TK
gene. These observations have to be considered in calculating the rate
of de novo-appearing FIAUr clones per cell per day. Based
on a 24-h generation time of the cells, the observed frequency reflects
a 3-day delay. Thus, the relevant cell number was eight times smaller,
and consequently the rate of de novo-appearing FIAUr clones
per cell per day was 3.6 × 102 to 3.8 × 102.
After the "run" procedure, FIAUr ES colonies were again
screened by PCR (primers 17F-H and 17+R) for the presence of the
HCHWA-D mutation. Revertants that had lost the selection cassette but retained the mutation were obtained from only 1 of the 12 hit clones
subjected to counterselection. But, 24 to 31% of the FIAUr
colonies which derived from this single clone (no. 110) were PCR
positive (Fig. 3B). Such positive ES colonies, as well as the precursor
clone, were further characterized by Southern analysis. Cellular
genomic DNA, restricted with endonucleases HindIII and AatII, was hybridized with the intron 16-specific probe 4 (Fig. 1). While a single band at 5.6 kb was detected on wild-type DNA, an additional 4.6 kb band was found on DNA from targeted mutated hit-and-run clones, indicating the presence of the HCHWA-D allele by
the mutant-specific AatII site on exon 17 (Fig.
5). Unequal intensities of wild-type and
mutant bands are found, as in the targeted-in cell analysis, due to the
presence of various amounts of feeder cell DNA. By Southern blot
analysis, the 5' position of the HCHWA-D mutation on the partial
genomic duplication of clone 110 was confirmed, as had already been
shown by PCR, making this clone unique among the hit clones tested
(Fig. 4A). Together with the same two bands as those for the
hit-and-run clones, representing the wild-type allele and the HCHWA-D
mutation on the 5' site of the partial duplication, a third band at 5.3 kb was detected, indicating that exon 17 on the 3' site is of the wild
type (Fig. 5). Since all PCR-positive FIAUr clones were
verified as pure HCHWA-D mutants, the frequency of intrachromosomal
recombination leading to de novo-generated mutants per day and cultured
cell was 1.1 × 102 to 1.2 × 102
for the run of clone 110.
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FIG. 5.
Characterization of ES cells produced by the hit-and-run
procedure. Verification of PCR-positive clones of the hit-and-run
procedure by Southern blot analysis. Aliquots containing 5 µg of
genomic DNA from ES cell clones (cultured on PMEFs) were double
digested with AatII/HindIII and hybridized
with intron 16-specific probe 4 after electrophoretic separation
through 2% agarose gels and transfer to a nitrocellulose membrane. A
single band at 5.6 kb was obtained from wild-type ES cells (wt) while
an additional band at 4.6 kb was obtained from hit-and-run clones for
the targeted allele carrying the HCHWA-D mutation. From the hit clone
110, which served as precursor for these positive clones, a third band
at 5.3 kb was detected, indicating the absence of an HCHWA-D mutation
on the 3' site of the partial genomic duplication. Numbers at tops of
lanes are clone designations.
|
|
| |
DISCUSSION |
Introduction of subtle mutations into the mammalian genome is
still a difficult task. Especially these days, however, it has become
increasingly interesting to subtly modify proteins in animals for
functional analysis. In this study, an isolated mouse genomic -APP
fragment was used for introduction of SFAD and HCHWA-D mutations into
the genome of mouse ES cells by homologous recombination by two
different methods. Both procedures had several common features. (i) All
targeting vectors contained the same 11-kb-long homology to the
cellular -APP gene. (ii) The same selection cassette was used to
control the homologous recombinations by G418 selection for the
presence of the neomycin gene in the first step and by FIAU selection
for the absence of the TK gene in the second step. Finally, (iii) the
experimental conditions for transfection and selection of ES cells were
standardized. These fixed parameters, together with defined differences
between the two methods, allowed interpretation of the results with
respect to some general mechanisms related to gene targeting by truly
homologous recombination.
Genomic DNA and the selection cassette were arranged differently on the
two types of vectors. Whereas the replacement vectors for the in-out
approach were linearized at the ends of the -APP region, the
insertion vector for the hit-and-run procedure contained a
double-strand break within the genomic fragment. Independent of the
vector types, the frequencies for ES cells that were either targeted in
or hit and had integrated the desired mutation into their genome after
the first homologous recombination were nearly identical. Also the
ratios of homologous recombination to random integration of targeting
vectors of the two methods were comparable. These results confirm that
mainly the overall extent of homology (11 kb long in these experiments)
and not the arrangement of the homologous regions determines the
probability of recombination in mammalian cells, and the frequencies
fit well with predicted values (7).
Characterization of targeted-in ES cells revealed correctly replaced
-APP loci with the targeted mutation and the intron-located selection cassette; no vector repeats were found at the modified -APP locus. In contrast, hit ES cell clones had frequently
integrated not a single copy of the insertion vector, but also actually
contained site-directed integrations of tandemly arranged
(head-to-tail) vector repeats (42% of all hit clones). One explanation
for this difference between the hit and targeting-in steps might be a
different capacity for self-assembly of insertion and replacement
vectors. Only head-to-tail arrangements of insertion vectors, as found in several hit clones, resemble at the junction between two vectors the
naturally occurring chromosomal structure of the -APP gene. Such a
structure might be more stable than artificial combinations of other
vector arrangements. The observed multicopy inserts might thus
originate from homologous recombinations between the targeted locus and
preassembled insertion vector repeats.
The second part of both procedures, i.e., the run or targeting out, was
more complex than the first step. A major issue was a high spontaneous
wild-type reversion rate of targeted-in or hit clones when they were
subjected to FIAU counterselection. The number of FIAUr
clones was more than 2 orders of magnitude greater than expected for
targeting out, and ES cells which were mock transfected for targeting
out showed the same high frequency of wild-type reversion. For the run
procedure as well, high frequencies of spontaneous wild-type reversion
were found. These reversion rates were even 10 times higher than for
targeted-in clones. Within each group, however, the reversion
frequencies of individual targeted-in or hit clones were similar. The
higher reversion frequency of hit clones compared to that of
targeted-in clones suggests different mechanisms for the two
procedures. In both cases, dominating wild-type reversion cannot be
explained by a defective TK gene, silencing of the targeted locus, or
insufficient FIAU selection, since the TK gene was physically lost
together with the targeted mutation. Loss of the entire targeted allele
cannot be excluded. However, this might at best explain the
less-frequent wild-type reversion in the targeting-in-out procedure,
since the frequency of random chromosomal aberrations should not depend
on the targeting procedure. Similarly unlikely is the existence of few
wild-type ES cells, which might have survived the initial neomycin
selection by metabolic cooperation with mutant cells and then have
reappeared as wild-type clones after FIAU selection. Otherwise, a
broader distribution of the reversion frequencies for individual clones
would be expected, regardless of the method. If the high reversion rate
was caused by metabolic cooperation, the number of wild-type cells
surviving the first selection step might be reduced by simple changes
of the experimental procedures like culturing ES cells at lower
densities. This is unlikely, and thus a reduction of the wild-type
reversion rate is probably hard to achieve. Finally, in the
targeting-out step, the electroporation itself may have directly
induced the reversion to wild-type cells, possibly by an SOS response,
as was suggested previously (18).
Despite the observed high frequency of wild-type reversion, pure mutant
ES cells without a selection cassette were generated by the hit-and-run
procedure. However, only 1 of 12 hit clones resulted after the run in
desired targeted mutant cells. For the other hit clones, only wild-type
reversion was found although the rates of cells surviving FIAU
selection were similar for all hit clones. A high percentage (24 to
31%) of FIAUr cells originating from this particular clone
contained the desired mutation, suggesting that the extremely frequent
spontaneous wild-type reversion in the hit-and-run procedure (3.6 × 102 to 3.8 × 102 per cell per day)
happens because of intrachromosomal recombination between the
duplicated -APP sequences in the genome of hit ES cells. The genomic
structure of the targeted locus of this particular hit clone was
different from that of the rest. Among the seven hit clones with a
single-copy integration of the targeting vector, it was the only one
which contained the HCHWA-D mutation at the 5' site of the partial
-APP duplication. All other hit clones either had the HCHWA-D
mutation at the 3' site or contained a multiple vector integration. An
increased probability for crossovers close to the double-strand break
has been reported for integration of insertion vectors by homologous
recombination (8). This could have triggered generation of
more hit clones with the HCHWA-D mutation at the 3' site of the
duplication in the present study. Yet, why after intrachromosomal
recombination the mutation preferentially persisted in the genome when
located at the 5' site of the duplication is unclear. One reason might
be a translocation of the HCHWA-D mutation from 3' to 5' after the
integration, bringing the mutation into an environment of correctly
methylated DNA sequences which might be less susceptible to loss
through intrachromosomal recombination. Alternatively, the
recombination machinery might be coupled to DNA replication and induce
an orientation-specific preference for maintenance of the 5' part of
the duplication in the genome.
The location of the mutation on the intermediate genomic duplication in
hit clones is very crucial for keeping the mutation in the genome after
loss of the duplication by intrachromosomal homologous recombination
and thus should be considered in optimization strategies of the
hit-and-run procedure. Screening a broad range of hit clones with
different genomic patterns, i.e., with the mutation at either side of
the duplication, should be preferred over intense screening of clones
from the same hit origin. A high frequency of intrachromosomal
recombination resulting in mutant run clones might then be expected for
some hit clones. Moreover, both approaches can be optimized by
increasing the efficiency for homologous recombination itself.
According to Deng and Capecchi (7), the maximal frequency of
homologous recombination obtained with isogenic DNA of more than 14 kb
is 10 times higher than for the 11-kb nonisogenic genomic -APP
fragment used in this study. Such an increased frequency might then be
sufficient for identification of, e.g., targeted-out cells with subtle
mutations among the large number of wild-type revertants.
In conclusion, by use of the targeting-in-out approach, only SFAD
mutant ES cells with a remaining selection cassette in intron 17 were
obtained, while pure HCHWA-D mutant ES cells were isolated after the
hit-and-run procedure. This second approach turned out to be very
efficient. From one intermediate clone containing the partial genomic
duplication, ~30% of the ES cell clones analyzed after
counterselection were pure HCHWA-D mutants. Finally, the hit-and-run
technique, with such high efficiency, has the advantage of being
independent of a second transfection and leads to pure mutants without
leaving behind any sequence in the genome.
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-Amyloid Precursor Protein Gene of Mouse Embryonic Stem
Cells: a Comparison of Homologous Recombination Methods
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Abstract
Introduction
