|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 51, 36585-36591, December 17, 1999
From the We investigated whether mouse short interspersed
elements (SINEs) could influence the recombination frequency of foreign
DNA. Vectors harboring a reporter gene in combinations of SINEs B1 and/or B2 or a portion of long interspersed element-1 were prepared and
tested in vitro by a colony assay using HC11 murine mammary epithelial cells and in vivo by microinjection into
fetilized mouse eggs. In transfected HC11 cells, the number of colonies surviving G418 selection increased by 3.5-fold compared with control when the reporter was flanked by fused B1-B2 sequences. Similar results
were obtained from microinjection study; in fetuses 11.5 days post
coitum, transgene positives in control and SINE-flanked vectors were 16 and 53%, respectively. Individual B1- and B2-harboring vectors showed
equivalent activities with each other, as determined by the colony
assay (2.8-fold versus 3.2-fold compared with control). We
determined the contribution of homologous recombination to the
SINE-mediated increase in integration frequency through a polymerase
chain reaction-based strategy; in more than half of embryos transgenes
underwent homologous recombinations involving B1 sequences. These
results demonstrate that the SINE sequences can increase the
integration rate of foreign DNA and that such an increase is most
likely due to the enhancement of homologous recombination.
Mammalian genomes contain both unique and repetitive DNA
sequences. In addition to the highly abundant satellite DNA, which occurs in tandem arrays mostly clustered in heterochromatic regions, the genome also contains repeated sequences that are interspersed among
single-copy sequences of euchromatic regions, such as the short
interspersed repeated DNA elements
(SINEs)1 and long
interspersed repeated DNA elements. Many of these repetitive DNA family
members are present in extremely high copy numbers, more than
105 copies/haploid genome (reviewed in Refs. 1 and 2).
Both B1 (130 bp) and B2 (190 bp) sequences are murine SINEs with
unknown functions (3). From 130,000 to 180,000 copies of the B1 repeat
and from 80,000 to 120,000 copies of the B2 repeat are present in the
mouse genome scattered on all chromosomes (4). Kramerov et
al. (5) reported that members of the B1 family were nearly
identical with one another. Individual members of the B2 family also
show a high degree of homology, displaying only 3-5% deviations from
the B2 consensus sequence (3). The presence of so many short homologous
sequences throughout the mammalian and higher eucaryotic genomes has
implications in genetic recombinations (1, 6). There have been several
reports about the participation of SINE equivalents in homologous
recombinations; most of these data came from analyses of
disease-related genes, such as the globin gene and low density
lipoprotein receptor genes. Various DNA clones obtained from subjects
with thalassemia, hereditary persistence of fetal hemoglobin, or
familial hypercholesterolemia were shown to undergo deletions or
insertions by Alu-Alu homologous recombinations or illegitimate
recombinations between Alu family members and unrelated sequences and
thus gave a clue of nonrandom rearrangements (7). Also, the
duplications of the entire growth hormone gene (6), the lysozyme gene,
(8), and a part of exons of the low density lipoprotein receptor gene
(9) in evolution were proposed to have occurred via Alu-Alu
recombinations. These results suggest that the number of recombinations
involving Alu-like members is much higher than expected (1) and that
these sequences, in particular, serve as hot spots for recombination
events (6, 10).
The introduction of exogenous DNA into mammalian cells has become an
increasingly important procedure. An efficient integration of delivered
DNA into the host genome is the most critical step in the whole
procedure of many biological applications, especially in areas such as
transgenesis and gene therapy. However, little information is available
about the actual molecular events involved in the integration process.
The integration phenomenon appears to be predominantly random and
nonhomologous (11, 12); thus, as far as there is no additional
machinery or modulatory elements to accelerate the process, the
integration of a foreign sequence into the genome might occur nearly by
chance. Such a passive integration pattern may also limit the
integration frequency. Therefore, the development of strategies that
could lead to an efficient, nonrandom integration of foreign DNA could
have a far reaching impact on areas where efficient gene transfer is a
crucial step.
To design a vector that can mediate a highly efficient integration of
foreign DNA, we searched for sequences that have a potential of
increasing the recombination frequency between the input DNA and the
host genomic loci. Because the SINE sequences such as B1 and B2 and the
long interspersed repeated DNA element sequences such as L1 exist in
high copy numbers dispersed throughout the mouse genome, we examined
the possibility that these interspersed repetitive sequences could
efficiently guide an exogenous DNA into genomic loci. It was
demonstrated that the mouse SINE repetitive sequences, such as B1 and
B2, could significantly increase the integration frequency of flanking
sequences in cultured cells and fetuses and that the elevated
integration rate was mostly due to an increase in homologous
recombination between the exogenous and endogenous SINE repetitive sequences.
Vector Construction--
Mouse B1, B2, and L1 repetitive
sequences were PCR-amplified from the CBA genomic DNA template and
individually cloned into pGEM7zf and pSP73 vectors (Promega). The
primers used to amplify the B1 sequence were designed to contain
an XhoI or BamHI site at the 5' end, and their
sequences are 5'-TAACTCGAGCCGGGTGTGGTGGC-3' and
5'-TTGGATCCAGGGTTTCTCTGTG-3'. To amplify the B2 sequence
(GenBankTM accession number M31441), a set of
XhoI- or BglII-anchored primers,
5'-AAAGATCTGGTGAGATGGCTCAG-3' and
5'-ACACTCGAGCTGTCTTCAGACAC-3', were used. Amplified B1 (145 bp) and B2 (152 bp) fragments were eluted from agarose gel, digested
with relevant restriction enzymes, and then cloned in a fused form into
XhoI sites of pGEM7zf and pSP73 (pGB1/2 and pSB1/2,
respectively). The cloned repetitive sequences were identified by
sequencing (Sequenase, CLONTECH). The vector
p(B1/2)2 was constructed by subcloning the B1/2-containing AatII/EcoRI fragment of pGB1/2 into the
AatII and EcoRI sites of the pSB1/2 vector.
pB1/2
To make p(B1)2 Cell Culture, Transfection, and Southern Blot Analysis of
Selected Colonies--
An HC11 cell, a murine mammary epithelial cell
line, was cultured as described previously (16). Transfection was
performed by the calcium phosphate precipitation method. 10 µg of
test plasmid was cotransfected with 1 µg of a Rous sarcoma
virus-driven luciferase control plasmid. 48 h after the
transfection cells were collected, a portion of them was assayed for
luciferase activity to measure transfection efficiency, whereas the
rest was replated onto a 100-mm diameter dish (Nunc) in various
densities in a medium containing G418 (0.2 mg/ml, Life Technologies,
Inc.) to obtain stable transfectants. The G418-resistant,
Giemsa-stained colonies were counted after 10-14 days of selection and
normalized for transfection efficiency. For the luciferase assay a 100 µg of whole protein extract was added to 350 µl of assay buffer (25 mM glycylglycine, pH 7.8, 10 mM
MgSO4, 2 mM ATP), and the luciferase activity
was measured using a luminometer (Berthold model Lumat, Germany) that
injects 100 µl of a 0.05 mM luciferin (sodium salt,
Sigma) solution into the reaction vessel. For Southern blot analysis
G418-resistant colonies were selected individually using a cloning
cylinder (Sigma), expanded into large populations, and harvested to
prepare genomic DNAs. 10 µg of genomic DNA was digested with
HindIII, which cuts the vectors only once,
size-fractionated, and transferred to a nylone membrane. The resulting
blot was hybridized with a radiolabeled 1.9-kb SacI fragment
of p Manipulation and Culture of Mouse Embryos--
The procedures
for the manipulation of embryos were previously described (17).
4-5-week-old BCF1 mice were superovulated by intraperitoneal
injections (48 h apart) of serum gonadotrophin from a pregnant mare
(Folligon, Intervet) and human chorionic gonadotrophin (Chorulon,
Intervet) and mated with 8-week-old males of the same strain.
Microinjection was performed 24 h after human chorionic
gonadotrophin injection. The plasmid DNAs used in the microinjection
were linearized by digestion with ScaI and purified through
Elutip-d (Schleicher & Schuell). Microinjected eggs were allowed to
develop in vitro into blastocyst-stage embryos in M16 media
(Sigma) or transferred at 2-cell stage to pseudopregnant ICR foster
mice to obtain 11.5-dpc fetuses. PCR-based Detection of Transgenes and Homologous Recombination
Events--
To detect transgenes in blastocysts the embryos were
individually transferred to a 0.5-ml PCR tube containing embryo lysis buffer (19) and incubated at 50 °C for 30 min, boiled at 95 °C
for 15 min, and then subjected to PCR. To analyze fetuses they were
digested with proteinase K in an extraction buffer (10 mM Tris-Cl, pH 8.0, 0.1 M EDTA, pH 8.0, 0.5% SDS, and 20 mg/ml pancreatic RNase) and genomic DNAs were prepared as described
previously (20). The PCR primers used were
5'-GCTTGGGTGGAGAGGCTATTCG-3' and 5'-GTAAAGCACGAGGAAGCGGTCAGCC-3' and
were designed to amplify a 692-bp region of the NeoR
sequence in the transgene. PCR was performed for 30 cycles (for fetuses) or 40 cycles (for embryos) at 95 °C for 1 min, 55 °C for
1 min, and 72 °C for 1 min and for one cycle of final extension at
72 °C for 10 min. For the detection of homologous recombinants in
blastocyst embryos, fully expanded blastocyst embryos were treated as
mentioned above and subjected to PCR analysis using a primer set, P137
(5'-CCACCAAAGAACGGAGCC-3') and PB1d-3 (5'-AGACAGGGTTTCTCTGTGTA-3'). PCR was carried out for 30 cycles at 95 °C for 1 min, 58 °C
for 1 min, and 72 °C for 0.5 min and for one cycle of final
extension at 72 °C for 10 min. The PCR product was size-fractionated
by electrophoresis on a 1.75% agarose gel, transferred to a nylone membrane, and subjected to Southern blot analysis. The probe used was
500 bp of the PGK promoter region prepared from p Construction of Repetitive Sequence-containing Plasmids and
Characterization of Their Integration-mediating Activity--
B1
and B2 sequences were amplified from CBA genomic DNA by PCR and fused
with each other in the same orientation (named B1/2). The 3' A-rich
region that normally exists in endogenous copies was not included in
B1/2. We also amplified about 710 bp of L1 sequence encompassing part
of the 5'-untranslated region and ORF1, a segment of L1 reported to be
present at 4 × 104 copies/genome (21), and confirmed
its identity and orientation by digestion with PstI. Using
these elements, we constructed a series of vectors having various
combinations of the repetitive sequences (Fig.
1A). The rationale for
designing a fused construct was such that if the integration rate of
foreign DNA is largely determined by the frequency of collision between
exogenous and endogenous DNA elements sharing high sequence homology,
the fused heterodimer might be more beneficial in increasing the
integration rate than either of the monomers. All the test vectors
contained a PGK
HC11 murine mammary epithelial cell line was transfected with these
vectors and allowed to form stable transformants under the drug
selection; for later calibration a Rous sarcoma virus-driven luciferase
vector was included as an internal standard. After 10-14 days of
selection, the surviving colonies were stained with X-gal or Giemsa,
and the number of colonies was counted. As shown in Fig. 1B,
the number of colonies was reproducibly higher in repetitive
sequence-containing vectors. Compared with the p
To estimate the copy number of transgenes genomic DNAs from each of
independent G418-resistant clones transfected with the ScaI-linearized p Detection of Transgenes in 11.5-dpc Mouse Fetuses Microinjected
with p
BCF1 mouse eggs were microinjected with linearized
p(B1/2)2 Contribution of Individual Components of the Fused B1/B2 Sequence
to the Increase in Integration Frequency--
Although both B1 and B2
sequences belong to the mouse SINE family, they apparently do not share
any evolutionary relationships. Because they are not physically
associated with each other in the mouse genome, the contribution of
each element to the enhancement of flanking DNA integration could be
significantly different if the process is dependent on the DNA sequence
rather than on the copy number of the repetitive sequence in the genome.
To determine which of the two SINE members played a dominant role in
increasing the integration rates of foreign DNA, two vectors containing
either monomeric B1 or B2 sequence at both sides of the Identification of Homologous Recombination Events in
(p(B1d)2
With the integration-enhancing ability of B1d proven, we
next went on to analyzing the nature of the B1d-mediated
integration events in developing embryos. For this, microinjection was
performed with p
Of the 75 blastocyst embryos injected with linearized
p(B1d)2
In conclusion, these results demonstrated that repetitive sequences
such as B1 and B2 could substantially improve the integration frequency
of flanking foreign DNA and that such an increase occurred through an
enhancement in the homologous recombination events between the incoming
and resident repetitive sequences.
In this paper we presented evidence that the B1 and/or B2
repetitive sequences can mediate highly efficient integration of flanking genes into the genome of both differentiated HC11 cells and
developing mouse embryos. In an accompanying paper the integration frequency of the SINE-flanked vector has been examined in mouse preimplantation embryos.2 In
that report, the B1/B2-flanked vector was microinjected in a linear or
covalently closed circular form, and it showed high The ability of human Alu repeats to promote homologous recombination
has been investigated in several cases
(27-30). In those reports, the effects of Alu sequences on the
homologous recombination events were shown to be controversial; some
reported to be efficient and others indistinguishable from entirely
random recombination. The inconsistency might, in part, derive from the
absence of an assay to detect the homologous recombination events
easily and directly. The homologous recombination events were detected
by cloning integration sites on the genome followed by clone mapping and sequencing (27) or, less directly, by examining the colonies surviving the G418 and gancyclovir selection (29). The former is a
direct but labor-intensive and time-consuming method and thus could not
examine an enough number of recombination events, whereas the latter
could not exclude the possibility of Alu-involved illegitimate
recombinations in the doubly selected colonies. Our results
demonstrated that rodent SINE sequences, such as B1 and B2, could
consistently increase (about three times) the integration rates of
SINE-flanked vector in various experimental systems, such as cultured
HC11 cells, preimplantation embryos2 and 11.5-dpc fetuses.
Moreover, using the Hord-PCR method, the homologous recombination
events could be easily and definitively verified at the molecular level.
It has been suggested that the sequence of Alu repeat diverged too much
among the family members or the sequence length of Alu repeat (300 bp)
is too short to promote species-specific recombination in mammalian
cells (28, 29). Waldman and Liskay (31) have shown that it is the
length of uninterrupted homology that is important for efficient
integration but not the overall homology. p(B1d)2 The strong recombinational propensity of the SINE sequences has played
a significant role in the evolution of the mammalian genome. In fact,
members of the Alu repetitive DNA family have been implicated in many
different recombination events and thus can serve as "hot spots"
for recombinational events. However, within the life of an organism
these recombinational processes may be occurring only rarely because of
the high topological constraints exerted on the cellular genome (33).
In contrast, the B1 and B2 repetitive sequences placed on free-floating
transgenes are free from such constraints and thus may be easily
brought into close proximity to genomic cognates by diffusion,
resulting in an increased chance of recombination.
The expression frequency of a transgene, in addition to the integration
rate, is considered to be one of the most important factors in
transgenesis. The repetitive sequences B1 and B2 may help increase the
expressivity of the transgene by guiding the integration of flanking
genes into a transcriptionally favorable region in the host genome. In
fact, as resolved by the chromosomal painting with the B1 or B2
sequence probe, both B1 and B2 sequences are known to be preferentially
clustered in the R bands (34). Genes in R bands are known to replicate
early in the S phase, and virtually all of the widely expressed
housekeeping genes map to these chromosomal regions (35, 36). Because
the major proportion of the transgene integration mediated by the B1
and/or B2 sequences occurred through the homologous recombination at
the corresponding genomic loci according to our results (Fig. 7), it is
most likely that the transgenes are located at euchromatic regions and
have a high probability of being expressed properly. Therefore, the mammalian germline transformation system involving the SINE sequences has dual benefits; an increased frequency of transgenesis and an
improved expressivity of the transgene. In fact, as described in our
another report, the p(B1/2)2 It is generally accepted that the frequency of random integration into
a genome is highly affected by the sequences of transgene ends (26,
37). In measuring the integration efficiency of SINE-flanked vectors by
the microinjection experiment we excluded such potential bias by
linearizing both control and SINE-tagged vectors with the
ScaI restriction enzyme, which cuts both plasmids once
within the ampicillin-resistant gene leaving the repetitive sequences
positioned internally in the vectors. Therefore, the free ends of both
linearized vectors should be identical in the nucleotide sequence and
the frequency of illegitimate recombination is expected to be similar
in both vectors.
Finally, we are currently establishing an experimental protocol to
detect mouse embryos that have transgenes already integrated into their
genome at the preimplantation stage by applying the Hord-PCR method to
isolated blastomeres; when properly established, this method is
expected to allow the preselection of the transgene-positive embryos
that have a high propensity of expressing the transgene by virtue of
its integration into the B1 or B2 loci. Furthermore, this method may
find a highly valuable application in the production of transgenic
domestic animals, such as goats, sheep, and cows, where the application
of transgenic technology is limited mainly because of their long life
cycles and high cost.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: P.O. Box 115, Yusong, Taejon 305-600, South Korea. E-mail:
leekk@kribb4680.kribb.re.kr.
2
Y.-K. Kang, J. S. Park, C.-S. Lee, Y. I. Yeom, A.-S. Chung, and K.-K. Lee, submitted for publication.
The abbreviations used are:
SINE, short
interspersed element;
LINE, long interspersed element;
dpc, days post
coitum;
PGK, phosphoglycerate kinase;
PCR, polymerase chain reaction;
Efficient Integration of Short Interspersed Element-flanked
Foreign DNA via Homologous Recombination*
§,,,,¶
Animal Developmental Biology
Research Unit, Korea Research Institute of Bioscience and
Biotechnology, Taejon 305-600, South Korea and the
§ Department of Biological Sciences, Korea Advanced
Institute of Science and Technology, Taejon 305-701, South Korea
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
geo, p(B1/2)2
geo, and pgeo were prepared by
inserting a 4.7-kb XhoI/HindIII fragment of the
pPGKgeobpA (13) vector into the SalI and
HindIII sites of the pSB1/2, p(B1/2)2, and pSP73
vectors, respectively. A 710-bp L1 sequence spanning the
5'-untranslated region through the middle of the ORF1 of the L1
sequence (14) was amplified by PCR using a set of
XhoI-anchored L1-specific primers
(5'-CTGCCTCGAGTCTGGTTCGAACA-3' and
5'-GTGTCCTCGAGTACCTGGATGTT-3') and cloned into the
XhoI site of pSP73 and pGEM7zf vectors (pSL1 and pGL1,
respectively). The pGL1 vector was restricted with
AatII/EcoRI, and the resulting L1 fragment was
subcloned into the corresponding enzyme sites of the pSL1 vector to
generate p(L1)2. The pL1geo and p(L1)2geo
vectors were constructed in a similar way.
geo, p(B2)2geo and
p(B1d)2geo vectors, pSP73 was digested with
BglII and filled with Klenow enzyme. The linearized vector
was added with d(T) to their 3' ends using Taq polymerase to
prepare a kind of T-vector (15), into which a B1, B2, or deleted B1
(B1d) monomer was inserted (pSB1, pSB2, or
pSB1d). The B1d sequence was amplified
like the B1 sequence but using another B1 3' primer,
5'-AGCCCTAGCTGTCCTGG-3'. The pSB1, pSB2, and pSB1d
vectors were digested with PvuII, added with d(T) in the
same mannner as described above, and inserted once again with B1, B2, or B1d to make p(B1)2, p(B2)2, and
p(B1d)2 vectors. The orientations of the
repetitive sequences were confirmed by restriction digestions with
BamHI, BglII, and XhoI. The 4.7-kb
PGKgeo fragment was inserted into the
HindIII/SalI sites of these vectors to generate
p(B1)2geo, p(B2)2geo, and p(B1d)2geo vectors.
geo vector spanning a part of the LacZ and NeoR
gene, washed, and exposed to an x-ray film according to the standard procedure.
-Galactosidase staining of embryos
was carried out as described (18). Briefly, the embryos were rinsed
with phosphate-buffered saline, fixed in 2% paraformaldehyde and
0.02% glutaraldehyde in phosphate-buffered saline for 30 min (for
blastocysts) or 1 h (for fetuses). Staining was done for 3 h
(blastocysts) or overnight (fetuses) at 30 °C in a solution containing 0.1% X-gal, 2 mM MgCl2, 5 mM EGTA, 0.2% Nonidet P-40, 5 mM
K3Fe(CN)6, and 5 mM
K4Fe(CN)6·6H2O. Stained samples
were stored at 4 °C in phosphate-buffered saline.
geo by
HindIII/BamHI digestion.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
geo reporter gene. We analyzed the integration
frequency of these constructs by a colony assay, which measured the
number of G418-resistant colonies (22, 23).
View larger version (22K):
[in a new window]
Fig. 1.
Schematic diagrams of reporter constructs
containing various mouse repetitive sequences. A, the
reporter gene fragment, represented as a thick arrowed line,
consists of a PGK-1 promoter (indicated as PGKp), the coding
sequence of the Escherichia coli LacZ gene fused in frame
with the neomycin resistant gene (NeoR), and a
poly(A) signal sequence of the bovine growth hormone (bGH)
gene. Thin arrows represent the transcriptional direction of
the B1, B2, and L1 repetitive sequences. The 1.9-kb SacI
fragment used as the probe for Southern blot analysis is indicated.
B, colony assay with five kinds of vectors using HC11 cells.
After 12 days of selection the surviving colonies were counted after
Giemsa staining. Values shown have been corrected for the transfection
efficiency of the luciferase control vector and represent the sum from
four independent experiments. The total number of colonies counted was
3012.
geo control, the
colony number increased by about 1.8-fold when the geo sequence was
flanked with a single copy of a fused B1/2 sequence and by about
3.5-fold when the geo sequence was entrapped within a pair of B1/2.
A significant level of increase in integration frequency was also
observed when one or two copies of the L1 sequence was used as the
source of the repetitive sequences. These results suggest that the
repetitive sequences, especially the B1/2 sequence, positively
influenced the integration rate of a flanking transgene. The B1 and B2
sequences used to prepare the B1/2 in this experiment were sequenced
and compared with the previously reported ones (Fig.
2); the 130 bp of B1 sequence showed 90%
homology with the consensus sequence reported by Maraia (24), and the
135 bp of B2 sequence shared 92% homology with the B2 sequence
reported in GenBankTM (accession number M31441).
View larger version (20K):
[in a new window]
Fig. 2.
Sequence comparison of B1 and B2 elements
used in this experiment with the reported sequences. The 130 bp of B1 sequence used in this experiment had a 90% homology with the
previously reported B1 sequence (24), whereas the 135 bp of B2 sequence
showed a 92% homology with the sequence reported in
GenBankTM (accession number M31441). Dots
represent identity, and dashes represent missing
nucleotides.
geo or p(B1/2)2geo vector
were subjected to Southern blot analysis after digestion with
HindIII, which cuts the vector only once. When
hybridized with a 1.9-kb NeoR sequence as the probe (Fig.
1A), about 49% (18/37) of the p(B1/2)2geo clones turned out to carry only one or two copies of transgenes, whereas about 28% (5/18) of the pgeo clones showed one or two copies of transgenes.
geo and p(B1/2)2geo Vectors--
The results
of colony assay represent the combined consequences of the integration
and subsequent expression of the exogenous gene. However, there could
be colonies that did not survive the drug selection because of
inefficient expression of the NeoR gene, even though they
had the transgenes integrated in the genome. To assess the effect of
B1/2 on the integration efficiency alone and to clarify whether the
SINE-mediated integration and expression is maintained through the
later stage of mouse development, we evaluated the integration
frequency mediated by B1/2 in an in vivo system by
microinjecting the B1/2-containing recombinant DNA into the pronuclei
of fertilized mouse one-cell embryos.
geo or pgeo DNA and transferred to
pseudo-pregnant ICR mice. At 11.5 dpc the fetuses were isolated,
stained with X-gal, and analyzed for the presence of transgenes by PCR
and Southern blot analysis. The stage of 11.5 dpc was chosen because it
is one of the most critical stages in the murine development that are
associated with major changes in the developmental pattern and thus the
gene expression pattern (25). As shown in Table
I, 4 of 25 fetuses (16%) from pgeo
were identified as transgene-positive, while 17 of 32 (53.3%) from
p(B1/2)2geo turned out to carry the transgene. Among the
17 fetuses carrying the p(B1/2)2geo sequence, 8 stained
positive for -gal in isolated regions of the body, 2 showed an
unrestricted expression pattern over the whole body, and the remaining
7 (Fig. 3) did not show any -gal
expression. In the case of the pgeo control, two of the four fetuses
expressed -gal. Thus, the integration rates, not only in cultured
HC11 cells but also in fetuses, were shown to be significantly higher in the SINE-containing vector than in the control.
Transgene detection in 11.5-dpc fetuses produced from the
microinjection of one-cell eggs with pgeo or
p(B1/2)2geo vector
View larger version (19K):
[in a new window]
Fig. 3.
Southern blot analysis of fetuses produced
from the microinjection of one-cell eggs with
p(B1/2)2 geo vector.
Southern blot analysis was performed on 15 -gal-negative fetuses to
determine the possible presence of integrated transgenes. Only the
result for fetuses injected with p(B1/2)2geo vector is
shown. Genomic DNAs were prepared from the fetuses, digested with
HindIII, and analyzed by the Southern technique. The
probe used for hybridization is shown in Fig. 1A.
geo reporter
gene were constructed (Fig.
4A). In the newly prepared
p(B1)2geo and p(B2)2geo vectors, the
orientation of the B1 or B2 sequences relative to the reporter gene was
reversed compared with that of p(B1/2)2geo to examine
whether the orientation of the repetitive sequences could affect the
expression of the reporter gene and thus the integration frequency.
Colony assay was performed according to the above-mentioned procedure.
The result showed that each of the B1 and B2 monomer sequences can enhance the integration frequency to a similar degree (Fig.
4B). Moreover, the integration frequency of the B1/2 fusion
sequence was only slightly higher than those with either of the
monomeric sequences inserted in the reverse orientation. Thus, the
three constructs carrying the repetitive modules seemed to be
functionally equivalent in guiding the transgenes into the genome.
Considering the results of Figs. 1B and 4B, it
appears that the number of isolated repetitive sequence modules in a
vector is a critical variable in increasing the chances for these
sequences to collide with the endogenous homologs.
View larger version (22K):
[in a new window]
Fig. 4.
Determination of the contribution of
individual B1 and B2 sequences to the enhancement of foreign DNA
integration. A, schematic diagram of the reporter
constructs used to measure the contribution of individual B1 and B2
sequences to the integration rate. Note that the orientation of the
repetitive sequence elements in p(B1)2 geo and
p(B2)2geo is opposite to that in
p(B1/2)2geo. B, the result of colony assays
for the constructs outlined in A. HC11 cells were
transfected with each vector, cultured for about 12 days in the
presence of G418, and then stained with Giemsa. Values shown represent
the means from two independent experiments.
geo-injected Blastocyst Embryos by
Hord-PCR--
Because the only difference between the repetitive
sequence-flanked vector and the control vector was the presence of the repetitive sequences, it is reasonable to infer that the repetitive sequence was responsible for the increase in integration rates of
transgenes. One of the mechanisms underlying this increase could be the
enormous chance of homologous recombination of the incoming B1 or B2
sequence of the vector with the corresponding, high copy numbered
cellular homologs. To determine the nature of the integration events,
we prepared a vector that can test the sequence-dependent
(homologous) recombination events between the two homologs of B1
sequences. Basically, the construct was equal to
p(B1)2geo except for a deletion of a part (20 bp of B1
3'-terminal region) of the repetitive sequence
(p(B1d)2geo; Fig.
5A). Lehrman et al.
(9) reported that the examined recombinational breakpoints of Alu
sequence were mostly centered in the middle region, especially between
the two RNA polymerase III promoters. Therefore, the
p(B1d)2geo construct was designed to retain
the entire middle region of the B1 sequence, lacking the proximal part
only, so that the inherent recombination activity would not be
disturbed. The p(B1d)2geo vector has been
evaluated in vitro and in vivo for the integration capacity before it is applied for the analysis of B1-mediated integration events. In a colony assay, the
p(B1d)2geo was shown to be capable of
integrating at a frequency about two times greater than that of the
pgeo control vector (Fig. 5B). Also, 52% (36/69) of the
blastocyst embryos microinjected with linearized
p(B1d)2geo DNA were transgene-positive when
analyzed by a combined PCR and Southern blot procedure (Fig.
5C). These results once again demonstrate the high
integration-mediating capability of the B1d sequence. The
extra bands in the PCR-positive lanes (3, 5, 9, 18, 19, 27, and 29) in
Fig. 5C appear to be artifacts associated with the
positivity because it was also shown in the positive control.
View larger version (65K):
[in a new window]
Fig. 5.
Schematic representation of the
p(B1d)2 geo vector used
for the detection of homologous recombinants. A,
diagrammatic structure of p(B1d)2geo
construct specifically designed to detect homologous recombination
events at B1 loci. Note that partially deleted B1 3' region is
represented as faint stippled boxes. A set of primers, P137
and PB1d, used for the specific detection of clones that have undergone
homologous recombination is indicated. Locations of the Hord-PCR
product and the probe used in Southern blot analysis are also shown.
B, the result of colony assay for the construct outlined in
A. Values shown represent the mean from two
independent experiments. C, PCR and Southern blot analyses
of the blastocyst embryos microinjected with
p(B1d)2geo. About 700 bp of NeoR
region was amplified from the PCR. A total of 69 blastocyst embryos
were examined, and only 36 embryos are represented in this figure. The
arrow (Neo) indicates the position of amplified positive
band position. Faint bands of a proper size are also shown in
lanes 21, 23, and 31.
geo and p(B1d)2geo
vectors, and the eggs were cultured into the blastocyst embryos.
Individual embryos were analyzed by a PCR process designed to detect
only the homologous recombination event (named Hord-PCR; see Fig.
6). When homologous recombination occurs,
the B1d sequence near the reporter gene turns into an
intact B1 sequence by acquiring from cellular B1 sequence the truncated
20-bp sequence with which the primer B1-3-3' can anneal. The other
primer was designed to anneal with the 5' PGK promoter region, and
thus, with this primer pair, PCR of the homologous recombination
product should yield a 300-bp fragment consisting of a part of the PGK promoter and a part of the B1 sequence from homologous recombinants. The specific PCR product of the homologous recombinants was detected by
Southern blot analysis using a 500-bp PGK promoter sequence as the
probe. In Fig. 7, the extra bands
associated with PCR positivity can be seen in lanes 12,
20, 21, 25, and 27 as well
as in the positive control; only the results for 40 out of 75 samples
are shown.
View larger version (16K):
[in a new window]
Fig. 6.
The Hord-PCR strategy that can detect
homologous recombinants. The primers P137 and PB1d can prime the
amplification of a 300-bp DNA fragment only when the input DNA has
undergone homologous recombination with an intact endogenous B1
sequence. P137 was derived from the 5' region of the PGK promoter and
thus could prime the DNA synthesis regardless of the occurrence of
homologous recombination. PB1d, derived from the 3' region of intact B1
sequence and deleted from the p(B1d)2 geo,
cannot prime the DNA synthesis unless there has occurred a homologous
recombination event between the B1d sequence and the
endogenous B1 sequence.
View larger version (29K):
[in a new window]
Fig. 7.
PCR and Southern hybridization analyses of
preimplantation embryos microinjected with linearized
p geo or
p(B1d)2geo
vectors. Blastocyst embryos microinjected with pgeo or
p(B1d)2geo were subjected to PCR analysis.
The PCR products were size-fractionated and analyzed by the Southern
technique using the 300-bp probe described in the legend to Fig. 6. The
hybridization probe was prepared as described in the legend to Fig. 6.
The results for 40 out of 75 embryos microinjected with
p(B1d)2geo DNA are shown; those for
remaining 35 are not shown. A number of positive signals with varying
intensities, representing homologous recombination events, can be seen.
The signal in the 22nd sample has a unique pattern, i.e.
smaller size and no smearing, and therefore, it may not represent the
normal Hord-PCR product. Ps, p(B1)2geo DNA as
the positive control; N1 and N2, no template DNA;
N3 and N5, p(B1d)2geo;
C1-C10, blastocyst embryos injected with the pgeo
control vector.
geo DNA, about 21% (15/75) was
clearly shown to have undergone homologous recombination (Fig. 7).
Because there were two identical B1d sequences on the
vector and the Hord-PCR was applied only to a single B1d
sequence, this value might represent only half of the actual recombination frequencies, assuming that both B1d sequences
have a similar capability of recombination. Thus, considering the
frequency of the B1d-mediated foreign DNA integration
events as shown in Fig. 5C, it could be reasoned that more
than half of the p(B1d)2geo-injected
transgene-positive embryos had the transgenes integrated via the
homologous recombinations involving the B1 sequences.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal expression
rates (more than three times than the control) in either conformation.
Further, we demonstrated in the present work that a significant
proportion of the augmented integration rate is due to the homologous
recombination between the endogenous and incoming repetitive sequences.
geo, the vector used for the
detection of homologous recombination events in our experiment, carries
only 110 bp of B1 sequence and yet showed a homologous integration
frequency two or three times greater than the control. Moreover,
because of the sequence divergence among the family members, the
longest stretch showing perfect match with the B1 consensus in the
B1d sequence extends only 18 bp, which is less than the
homology length of 25 bp known to be sufficient for recombination (32). Therefore, our result demonstrated that a SINE repeat that is as short
as 110 bp in length and carries only an 18-bp stretch of complete
sequence homology (Fig. 2) could mediate an efficient integration of
flanking foreign DNA into the endogenous B1 loci.
geo-injected blastocyst
embryos showed much higher levels of -gal expression compared with
the pgeo-injected control embryos.2
FOOTNOTES
ABBREVIATIONS
-gal, -galactosidase;
Hord, homologous recombination detection;
bp, base pair(s);
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
kb, kilobase(s).
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Berg, D. E.,
and Howe, M. M.
(1989)
Mobile DNA
, pp. 593-637, American Society for Microbiology, Washington, D.C.
2.
Weiner, A. M.,
Deininger, P. L.,
and Efstratiadis, A.
(1986)
Annu. Rev. Biochem.
55,
631-661[CrossRef][Medline]
[Order article via Infotrieve]
3.
Krayev, A. S.,
Markusheva, T. V.,
Kramerov, D. A.,
Ryskov, A. P.,
Skryabin, K. G.,
Bayev, A. A.,
and Georgiev, G. P.
(1982)
Nucleic Acids Res.
10,
7461-7475 4.
Bennett, K. L.,
Hill, R. E.,
Pietras, D. F.,
Woodworth-Gutai, M.,
Kane-Haas, C.,
Houston, J. M.,
Heath, J. K.,
and Hastie, N. D.
(1984)
Mol. Cell. Biol.
4,
1561-1571 5.
Kramerov, D. A.,
Grigoryan, A. A.,
Ryskov, A. P.,
and Georgiev, G. P.
(1979)
Nucleic Acids Res.
6,
697-713 6.
Barsh, G. S.,
Seeburg, P. H.,
and Gelinas, R. E.
(1983)
Nucleic Acids Res.
11,
3939-3958 7.
Ottolenghi, S.,
and Giglioni, B.
(1982)
Nature
300,
770-771[CrossRef][Medline]
[Order article via Infotrieve]
8.
Cross, M.,
and Renkawitz, R.
(1990)
EMBO J.
9,
1283-1288[Medline]
[Order article via Infotrieve]
9.
Lehrman, M. A.,
Goldstein, J. L.,
Russell, D. W.,
and Brown, M. S.
(1987)
Cell
48,
827-835[CrossRef][Medline]
[Order article via Infotrieve]
10.
Jagadeeswaran, P.,
Tuan, D.,
Forget, B. G.,
and Weissman, S. M.
(1982)
Nature
296,
469-470[CrossRef][Medline]
[Order article via Infotrieve]
11.
Kato, S.,
Anderson, R. A.,
and Camerini-Otero, R. D.
(1986)
Mol. Cell. Biol.
6,
1787-1795 12.
Murnane, J. P.,
Yezzi, M. J.,
and Young, B. R.
(1990)
Nucleic Acids Res.
18,
2733-2738 13.
Friedrich, G.,
and Soriano, P.
(1991)
Genes Dev.
5,
1513-1523 14.
Loeb, D. D.,
Padgett, R. W.,
Hardies, S. C.,
Shehee, W. R.,
Comer, M. B.,
Edgell, M. H.,
and Hutchison, C. A. D.
(1986)
Mol. Cell. Biol.
6,
168-182 15.
Marchuk, D.,
Drumm, M.,
Saulino, A.,
and Collins, F. S.
(1991)
Nucleic Acids Res.
19,
1154 16.
Doppler, W.,
Groner, B.,
and Ball, R. K.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
104-108 17.
Hogan, B.
(1994)
Manipulating the Mouse Embryo: A Laboratory Manual
, 2nd Ed.
, pp. 226-252, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
18.
Sanes, J. R.,
Rubenstein, J. L.,
and Nicolas, J. F.
(1986)
EMBO J.
5,
3133-3142[Medline]
[Order article via Infotrieve]
19.
Krisher, R. L.,
Gibbons, J. R.,
Canseco, R. S.,
Johnson, J. L.,
Russell, C. G.,
Notter, D. R.,
Velander, W. H.,
and Gwazdauskas, F. C.
(1994)
Transgenic Res.
3,
226-231[CrossRef][Medline]
[Order article via Infotrieve]
20.
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, pp. 9.16-9.23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
21.
Edgell, M. H.,
Hardies, S. C.,
Loeb, D. D.,
Shehee, W. R.,
Padgett, R. W.,
Burton, F. H.,
Comer, M. B.,
Casavant, N. C.,
Funk, F. D.,
and Hutchison, C. A. D.
(1987)
Prog. Clin. Biol. Res.
251,
107-129[Medline]
[Order article via Infotrieve]
22.
Lin, H. J.,
Anagnou, N. P.,
Rutherford, T. R.,
Shimada, T.,
and Nienhuis, A. W.
(1987)
J. Clin. Invest.
80,
374-380
23.
Martin, D. I.,
Tsai, S. F.,
and Orkin, S. H.
(1989)
Nature
338,
435-438[CrossRef][Medline]
[Order article via Infotrieve]
24.
Maraia, R. J.
(1991)
Nucleic Acids Res.
19,
5695-5702 25.
Allen, N. D.,
Cran, D. G.,
Barton, S. C.,
Hettle, S.,
Reik, W.,
and Surani, M. A.
(1988)
Nature
333,
852-855[CrossRef][Medline]
[Order article via Infotrieve]
26.
McFarlane, M.,
and Wilson, J. B.
(1996)
Transgenic Res.
5,
171-177[CrossRef][Medline]
[Order article via Infotrieve]
27.
Wallenburg, J. C.,
Nepveu, A.,
and Chartrand, P.
(1987)
Nucleic Acids Res.
15,
7849-7863 28.
Shen, M. R.,
and Deininger, P. L.
(1992)
Anal. Biochem.
205,
83-89[CrossRef][Medline]
[Order article via Infotrieve]
29.
Watson, J. E.,
Slorach, E. M.,
Maule, J.,
Lawson, D.,
Porteous, D. J.,
and Brookes, A. J.
(1995)
Genome Res.
5,
444-452 30.
Pavan, W. J.,
and Reeves, R. H.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
7788-7791 31.
Waldman, A. S.,
and Liskay, R. M.
(1988)
Mol. Cell. Biol.
8,
5350-5357 32.
Ayares, D.,
Chekuri, L.,
Song, K. Y.,
and Kucherlapati, R.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
5199-5203 33.
Nicholls, R. D.,
Fischel-Ghodsian, N.,
and Higgs, D. R.
(1987)
Cell
49,
369-378[CrossRef][Medline]
[Order article via Infotrieve]
34.
Boyle, A. L.,
Ballard, S. G.,
and Ward, D. C.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
7757-7761 35.
Holmquist, G. P.
(1992)
Am. J. Hum. Genet.
51,
17-37[Medline]
[Order article via Infotrieve]
36.
Craig, J. M.,
and Bickmore, W. A.
(1993)
Bioessays
15,
349-354[CrossRef][Medline]
[Order article via Infotrieve]
37.
Chen, C. M.,
Choo, K. B.,
and Cheng, W. T.
(1995)
Transgenic Res.
4,
52-59[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Iiizumi, A. Kurosawa, S. So, Y. Ishii, Y. Chikaraishi, A. Ishii, H. Koyama, and N. Adachi Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting Nucleic Acids Res., November 1, 2008; 36(19): 6333 - 6342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Dodge, Y.-K. Kang, H. Beppu, H. Lei, and E. Li Histone H3-K9 Methyltransferase ESET Is Essential for Early Development Mol. Cell. Biol., March 15, 2004; 24(6): 2478 - 2486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. KAMINSKI, M. R. HUBER, J. B. SUMMERS, and M. B. WARD Design of a nonviral vector for site-selective, efficient integration into the human genome FASEB J, August 1, 2002; 16(10): 1242 - 1247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. de Vries and W. Wackernagel Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination PNAS, February 19, 2002; 99(4): 2094 - 2099. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |