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Blood, 1 July 2001, Vol. 98, No. 1, pp. 65-73
GENE THERAPY
Lack of neighborhood effects from a transcriptionally active
phosphoglycerate kinase-neo cassette located between the
murine  -major and -minor globin genes
Richard M. Kaufman,
Zhi
Hong Lu,
Rajesh Behl,
Jo M. Holt,
Gary K. Ackers, and
Timothy J. Ley
From the Departments of Pathology/Laboratory Medicine,
the Division of Oncology, Section of Stem Cell Biology, and the
Department of Biochemistry, Washington University School of Medicine,
St Louis, MO.
 |
Abstract |
For the treatment of  -globin gene defects, a homologous
recombination-mediated gene correction approach would provide
advantages over random integration-based gene therapy strategies.
However, "neighborhood effects" from retained selectable marker
genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the 6 position of
the mouse -major globin gene (6I) as a step toward
the development of a murine model system that could serve as a platform
for therapeutic gene correction studies. The marked -major gene can
be tracked at the level of DNA, RNA, and protein, allowing
investigation of the impact of a retained phosphoglycerate kinase
(PGK)-neo cassette located between the mutant -major and -minor
globin genes on expression of these 2 neighboring genes. Although the
PGK-neo cassette was expressed at high levels in adult erythroid cells,
the abundance of the 6I mRNA was indistinguishable from
that of the wild-type counterpart in bone marrow cells. Similarly, the
output from the -minor globin gene was also normal. Therefore, in
this specific location, the retained, transcriptionally active PGK-neo
cassette does not disrupt the regulated expression of the adult
-globin genes.
(Blood. 2001;98:65-73)
© 2001 by The American Society of Hematology.
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Introduction |
Since the molecular cloning and sequencing of the
globin genes more than 20 years ago, many investigators have explored
gene therapy as a potential mechanism for treating -globin gene
defects.1 The gene therapy approach that has been most
carefully studied makes use of retroviral vectors to introduce
-globin genes into hematopoietic stem or progenitor cells, where the
-globin gene randomly integrates into the host cell genome (reviewed
in 2). This approach has met with limited success for a
variety of reasons, including low efficiency of progenitor cell
transduction,3,4 variegation of expression of the
integrated -globin genes, and silencing of the integrated -globin
genes that occurs as a function of time.5,6 When the locus
control region was identified more than a decade ago, the core regions
of the hypersensitive sites were added to retroviral vectors to reduce
or eliminate the integration site-specific variegation of -globin
gene expression.7-9 Unfortunately, this change in the
design of these vectors led to instability in the viral genomes on
integration, which limited the usefulness of the locus control region
(LCR) in retroviral systems.7,8 Although improvements in
the system have recently been described using lentivirus-based vectors,
fundamental problems with regulated -globin gene expression
still exist.10 In addition, retroviral vectors necessarily
cause mutations at sites of integration, and the results of these
mutations in any given recipient cell cannot be predicted. The
mutagenesis caused by random integration remains a problem for most
gene therapy strategies.
Alternative approaches for the gene therapy of -globin disorders
include "gene correction"11,12 and homologous
recombination.13-15 Shesely et al13 first
described the production of a targeted mutation of the human -globin
locus with homologous recombination in mouse erythroleukemia (MEL)
cells containing a single copy of human chromosome 11. In that
experiment, the authors used a targeting vector that contained 4.7 kb
nonisogenic human DNA in the targeting arms. One integration event in
10 000 was shown to be homologous. More recently, using vectors
containing 2 to 3 kb isogenic DNA in each targeting arm and positive
selection only, we and others have shown that many vectors
homologously recombine 1% to 2% of the time.
An additional unexpected effect of homologous recombination has been
described by many laboratories retained selectable marker cassettes
can have strong effects on the expression of neighboring genes.16-24 Groudine et al16,17 first
reported that when selectable marker cassettes are targeted to the LCR
of the -globin gene cluster, the LCR appears to preferentially
interact with the selectable marker gene so that the expression of
downstream globin genes is dramatically reduced. "Neighborhood
effects" have also been described in many other multigene loci, and
the effect can be noticeable for hundreds of
kilobases.20-23 The neighborhood effect apparently has a
directional component in some loci,21 but general rules
for neighborhood effects have not yet been established.
The low efficiency of homologous recombination in MEL cells and the
powerful neighborhood effects of retained selectable marker genes in
the -globin locus suggested that homologous recombination-mediated gene correction of the -globin gene would not be useful. In this study, however, we created a subtle mutation in the 6 position of
the mouse -major globin gene (6I), a mutation
that resembles the S mutation in humans. This mutation
allows us to track the mutant gene at the level of DNA, RNA, and
protein. The mutant locus contains a transcriptionally active
phosphoglycerate kinase (PGK)-neo cassette between the
-major and -minor globin genes. Remarkably, the retained PGK-neo
cassette did not affect the output of either -globin gene,
suggesting that neighborhood effects may not limit the ability of
homologous recombination to be used as a gene correction strategy for
mutant -globin genes.
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Materials and methods |
Construction of the 6I targeting vector
The targeting strategy is depicted in Figure
1A. The targeting vector consists of a
6-kb left-targeting arm, a LoxP-flanked phosphoglycerate kinase I gene
promoter-driven neomycin resistance gene cassette (PGK-neo), and a
2.2-kb right-targeting arm. The left-targeting arm, containing a 6-kb
AvaI fragment spanning the mouse -major gene, was
isolated from a 110-kb 129/SvOla DNA-derived P1 clone (designated
1935), containing the entire mouse -globin cluster. The left arm was
subcloned into pGEM3zf+ (Promega, Madison, WI). Site-directed
mutagenesis of codon 6 (GCT ATC) was then performed using the
Quickchange mutagenesis kit (Stratagene, La Jolla, CA), and the
presence of the "6I" mutation was confirmed by
sequence analysis. The entire -major gene and flanking DNA were also
sequenced and were found normal. Using P1 1935 as the template, the
right-targeting arm was generated by polymerase chain reaction
(PCR) using the forward primer
5'-ACGTGGATCCACTGCGAGGCAGAGGCAGGC-3' and the reverse primer
5'-ACGTAAGCTTTATGCCTCAGTACAGGGGAT-3', which generates a
2.2-kb BamHI-HindIII fragment from sequences
located between 4131 and 6370 bp downstream from -major cap site.
The left arm, PGK-neo cassette, and right arm were cloned in the proper orientation in pCR 2.1 (Invitrogen, Carlsbad, CA), as shown in Figure
1A. No DNA sequences were removed from the targeted locus.
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| Figure 1.
Targeting strategy for generating the 6I
mutation.
(A) Top line depicts the structure of wild-type -major genomic
locus; the 6I targeting vector is shown in the middle.
The targeted allele with a retained PGK-neo cassette is depicted on the
bottom line. Locations of external probe 166 and internal probe 169 are
shown. (B) Comparison of DNA and amino acid sequences of mouse
-major globin and human globin genes. (C) Southern blot analysis
of genomic tail DNA derived from the progeny of 6I
heterozygous mutant mice. (left) DNA digested with XbaI and
hybridized with probe 166. (right) DNA digested with EcoRV
and BglII and hybridized with probe 169.
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Creation of 6I mice
Forty micrograms targeting vector was linearized by
ApaI digestion and electroporated into RW4 embryonic stem
(ES) cells (derived from a 129/SvJ mouse) as previously
described.19 G418-resistant ES clones were isolated and
screened by Southern blot analysis using the 3' external probe
designated 166. Two correctly targeted ES clones (83 and 140) were
confirmed by Southern blot analysis to contain the 6I
mutation. C57BL/6 blastocysts were then microinjected with both clones
and implanted into pseudopregnant Swiss Webster foster females to
generate chimeras, as previously described.19 Chimeric males were bred with wild-type 129/SvJ female mice, and germline transmission of the 6I mutation was confirmed by
Southern blot analysis.
RNA expression analysis
Total RNA from mouse bone marrow and peripheral blood was
isolated as described.25 Two micrograms total RNA from
each sample was reverse-transcribed into complementary DNA (cDNA)
exactly as previously described25 and analyzed by PCR (see
below). The relative messenger RNA (mRNA) expression of
6I and the wild-type -major gene was measured by a
real-time PCR assay using the TaqMan system (PerkinElmer, Norwalk, CT).
An exon 1 forward primer specific for either wild-type mouse
-major sequence 5'-TGGTGCACCTGACTGATGCT-3') or
6I sequence 5'-TGGTGCACCTGACTGATATC-3') was
used in combination with a universal exon 2 reverse primer
5'-CCATGGGCCTTCACTTTGGC-3'). The output from the wild-type -major
allele or the mutant 6I allele was measured using either
the wild-type primer set or 6I primer set, respectively.
The PCR reaction was performed in the presence of a DNA intercalating
dye, SYBR green. A PCR amplification profile was derived by recording
SYBR green fluorescence intensity, which was in linear relation to the
amount of PCR product formed, as a function of PCR cycle number. A
standard curve was generated by plotting the PCR cycle number at which
a reaction entered exponential amplification versus the amount of input
DNA and was then used for a determination of sample template
concentration.26
A semiquantitative reverse transcription (RT)-PCR assay was used to
measure murine peripheral blood -minor mRNA expression. A PCR primer
set specific for murine -minor gene was designed to span intron 1 (forward primer, 5'-GAGTCTGTTGTGTTGACTTG-3'; reverse primer,
5'-TTGTCCAGGTTTTTCAGGCCC-3'). The PCR product from the mouse -minor
cDNA measured 294 bp. A murine -actin gene primer set was included
as a control for RNA loading (forward primer,
5'-GCTGTATTCCCCTCCATCGTG-3'; reverse primer,
5'-CACGGTTGGCCTTAGGGTTCAG-3'), which generated a 265-bp PCR fragment.
The PCR reaction was performed in the presence of 20 µCi/mL
 -32P]dATP for 25 cycles. PCR products were resolved by
electrophoresis on tris borate EDTA (TBE)-5% polyacrylamide gels, and
radioactivity of individual bands was quantitated using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Relative -minor
mRNA expression was normalized for -actin mRNA abundance and
expressed as a percentage of wild-type expression.
Globin chain analysis
Triton-acid-urea-polyacrylamide gel electrophoresis (PAGE) was
used to quantitate globin chain composition of peripheral red blood
cells (RBCs) as previously described27 with minor modifications.
Hematopoietic progenitor cell assay
Mice were killed, and bone marrow cells were harvested from the
tibiae and femurs. Mononuclear cells were isolated using Histopaque 1077 (Sigma, St Louis, MO). Viable cells were quantified using trypan
blue staining. For analysis of colony-forming unit-granulocyte (CFU-G)
and colony-forming unit-granulocyte-macrophage (CFU-GM), the cells
were plated at a concentration of 50 000 cells/mL in Methocult M 3434 (Stemcell Technologies, Vancouver, BC, Canada), containing 10 ng/mL
recombinant murine interleukin-3 (rmIL-3), 50 ng/mL recombinant murine
stem cell factor (rmSCF), 10 ng/mL rhIL-6, and 3 U/mL recombinant human
erythropoietin (rhEPO), with or without 900 µg G418 (active)/mL
media. For analysis of burst-forming unit-erythroid (BFU-E), bone
marrow cells were plated in Methocult M3334 (Stemcell Technologies) at
a concentration of 200 000 viable cells/mL (with rhEPO at 3 U/mL),
with or without G418 at a concentration of 900 µg (active)/mL.
Colonies were quantified 8 days after plating.
Detection of neomycin phosphotransferase
Splenocytes, bone marrow, and peripheral blood cells were
harvested from wild-type mice and 6I homozygous mice,
cytospun onto coverslips, fixed in methanol, rehydrated in
phosphate-buffered saline (PBS), and blocked for 30 minutes in blocking
solution (0.1% Triton X-100, 1% bovine serum albumin in PBS).
Coverslips were incubated in rabbit anti-Tn5 Neo polyclonal antibody
(US Biological, Swampscott, MA) in blocking solution for 1 hour. The
coverslips were then washed twice in washing solution (0.1% Triton
X-100 in PBS), stained with fluorescein-conjugated goat antirabbit
secondary antibody for 1 hour, and washed twice in washing solution.
Cells were costained with phycoerythrin (PE)-conjugated monoclonal
antibodies directed against various mouse cell surface markers, as
previously described.28 Nuclear DNA was visualized with
4',6-diamidino-2-phenylindole (DAPI). Samples were viewed with a Nikon
Microphot-SA fluorescence microscope.
Neo protein was also detected using flow cytometric analysis. Bone
marrow cells were first stained with PE-conjugated monoclonal antibodies against various mouse cell surface markers (Ter-119, Gr-1,
or CD3). Then the cells were fixed in Cytofix/Cytoperm solution (Pharmingen, San Diego, CA) at 4°C overnight, washed twice in Perm/Wash buffer (Pharmingen), and incubated in Perm/Wash buffer containing rabbit anti-Neo antibody at 4°C for 30 minutes. The cells
were washed twice in Perm/Wash buffer, incubated with
fluorescein-conjugated antirabbit secondary antibody for 30 minutes,
and finally washed twice in Perm/Wash buffer. Dual-color flow
cytometric analysis was performed using a FACScan flow cytometer
(Becton Dickson, San Jose, CA), as previously described.28
Blood cell morphology and sickling test
Blood smears were prepared from oxygenated mouse peripheral
blood, followed by May-Grünwald-Giemsa staining. For the sickling test, 10 µL freshly collected blood was mixed with 10 µL 2% sodium metabisulfite solution on a slide and incubated under sealed coverslips at room temperature for 30 minutes to induce deoxygenation.
Hemoglobin solubility test
Solubility of hemolysates was studied by measuring
turbidity in a concentrated potassium phosphate buffer on
deoxygenation, exactly as previously described.29
Hematologic parameters
Peripheral blood was collected from the retro-orbital sinuses of
adult mice and analyzed using a Hemavet 850 system (CDC Technologies, Oxford, CT). Reticulocytes were stained with acridine orange and analyzed using a FACScan flow cytometer (Becton Dickson) as previously described.30
Hemoglobin oxygen-binding curve
The thin-layer optical device of Gill31 was used to
measure oxygenation isotherm as previously
described.32
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Results |
Design of the 6I mutation in the mouse -major
globin gene
In this study, we developed mice with a mutation in position 6 of
the mouse -major globin gene. This mutation, which changes GCT
(encoding Ala) to ATC (encoding Ile), was designed for several reasons
(Figure 1B). First, this mutation creates a novel EcoRV site
in the -major gene and serves as a genetic mark that can be followed
by restriction enzyme analysis. Second, the 6 Ala-to-Ile (6I) mutation should produce a mutant -globin chain
with an altered hydrophobicity, and it should be distinguishable from
wild-type -major globin using routine globin chain separation
methods. Third, the 6I mutation was predicted to produce
a murine hemoglobin that might polymerize on deoxygenation.
Creation of 6I mutant mice
We transfected RW-4 ES cells with the targeting vector shown in
Figure 1A. Four correctly targeted RW4 ES clones were identified out of
220 screened. Two clones (83 and 140) were arbitrarily selected for
further analysis and were confirmed on Southern blot to contain the
6I mutation. Both cell lines were then microinjected
into C57BL/6 blastocysts to generate chimeras. Both lines transmitted
the 6I mutation through the germline. Figure 1C shows a
Southern blot analysis of genomic tail DNA from 4 F1 littermates
produced by mating a chimera with a wild-type 129/SvJ female. Shown in
the left panel is a Southern blot of XbaI-digested tail DNA
derived from one wild-type 129/SvJ control mouse (lane 1) and the 4 F1 mice (lanes 2-5) and hybridization with the external probe 166. The
wild-type allele produced a 5.6-kb fragment. The mutant allele, containing an internal XbaI site within the PGK-neo
cassette, produced a diagnostic 3.8-kb fragment. Two of the mice (lanes 2 and 5) are heterozygous for the targeted allele. In the right panel,
the same group of DNA samples was hybridized with internal probe 169 after EcoRV/BglII double-digestion. The wild-type
fragment measured 3.4 kb. The 6I mutation created a new
EcoRV site that reduced the size of the hybridizing band to
2.7 kb. The presence of the predicted 2.7-kb EcoRV/BglII fragment confirmed that mice
represented in lanes 2 and 5 were heterozygous for the
6I mutation. Hybridization of blots with a neo-specific
probe revealed a single hybridizing band of the predicted size for each
enzyme tested; a single PGK-neo cassette was, therefore, integrated in the genome (data not shown). Heterozygous mice were bred with wild-type
129/SvJ mice, and heterozygous F2 mice were then intercrossed. The
ratio of
WT/WT:WT/6I:
6I/6I mice produced by these matings was
25:46:39.
Expression of adult -globin genes in 6I
mutant mice
The relative expression of the 6I and wild-type
-major genes was compared in bone marrow RNA obtained from
heterozygous 6I mice using a real-time PCR assay. In
this assay, an exon 1 forward primer specific for either wild-type
mouse -major sequence or 6I sequence was used in
combination with a universal exon 2 reverse primer. PCR reactions were
performed in the presence of a DNA intercalating dye, SYBR green. A PCR
amplification curve was derived in a real-time by plotting PCR product
fluorescence intensity ( Rn) on the y-axis versus PCR cycle number on
the x-axis. In Figure 2A, 10-fold serial
dilutions (100 pg, 10 pg, 1 pg, and 0.1 pg DNA) of pWT,
a plasmid containing the wild-type -major gene, were amplified using
either the WT primer set (reactions 1-4) or
6I primer set (reactions 5-8). Reaction 1 entered the
exponential phase of amplification at cycle 11. Decreasing the amount
of input template DNA resulted in a progressive delay in entry into the exponential phase. In contrast, the 6I-specific primer
did not amplify the wild-type -major template, as evidenced by a
lack of exponential rise in fluorescence (reactions 5-8), demonstrating
the specificity of this assay. Next, as shown in Figure 2B, 10 ng
plasmid DNA containing the mutant 6I
(p6I) or the wild-type -major sequence
(pWT) was used in a real-time PCR assay. Each plasmid
sample (or a control with no DNA) was amplified using either the
WT primer set (reactions 1-3) or 6I
primer set (reactions 4-6). Exponential amplification was only observed
when the templates were appropriately matched with their specific
primer sets (reactions 1 and 5 vs. reactions 2 and 4). Amplification
curves of reactions 1 and 5 were superimposable, suggesting that
wild-type -major and 6I sequences were amplified with
equal efficiency. Bone marrow RNA harvested from a 6I
heterozygous mouse or a wild-type control mouse was reverse-transcribed to cDNA and analyzed by real-time PCR (Figure 2C). The output from the
wild-type -major allele or mutant 6I allele was
measured, respectively, using either the WT primer set
(reactions 1 and 2) or 6I primer set (reactions 3 and
4). A reaction amplifying the wild-type peripheral blood cDNA with the
6I primer set was included as a negative control
(reaction 5). Amplification curves generated by reactions 1 to 4 were
indistinguishable from one another, indicating that the
6I allele was expressed at a level equivalent to that of
the wild-type -major gene. These experiments were repeated 3 times
with similar results.
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| Figure 2.
Targeted insertion of a PGK-neo cassette does not disrupt the regulated
expression of 2 neighboring murine -major and -minor globin
genes.
(A) Real-time PCR assay for the wild-type mouse -major genomic DNA.
PCR product fluorescence intensity (Rn) is plotted on the y-axis
versus PCR cycle number on the x-axis. Tenfold serial dilutions of
pWT, a plasmid encoding the wild-type -major gene,
were amplified using either the WT primer set (reactions
1-4) or 6I primer set (reactions 5-8). Plotting the PCR
cycle number at which the reaction entered exponential amplification,
versus the logarithm of the amount of input DNA, generated a standard
curve (not shown). (B) Ten nanograms plasmid DNA encoding the mutant
6I (p6I) or the wild-type -major
sequence (pWT) was used for real-time PCR. Each plasmid
sample or a no DNA control (No DNA) was amplified using either the
WT primer set (reactions 1-3) or the 6I
primer set (reactions 4-6). (C) Real-time PCR analysis of bone marrow
RNA. Bone marrow RNA was harvested from a heterozygous
6I mouse (WT/6I) or a
wild-type control mouse (WT/WT),
reverse-transcribed to cDNA, and analyzed by real-time PCR using either
the WT primer set (reactions 1-2) or 6I
primer set (reactions 3-4). A reaction amplifying the wild-type
peripheral blood cDNA with the 6I primer set was
included as a negative control (reaction 5). (D) Semiquantitative
RT-PCR analysis of peripheral blood RNA. Peripheral blood RNA was
harvested from homozygous 6I mice
(6I/6I) or wild-type control mice
(WT/WT), reverse-transcribed, and
analyzed by PCR using a -minor primer set or a -actin gene primer
set. Reactions were performed in the presence of
-32P]dATP and stopped before amplifications reached a
product plateau (not shown). Amplification products were resolved on
5% TBE-polyacrylamide gels and quantitated using a PhosphorImager. For
each experiment, the relative -minor mRNA expression was normalized
for -actin mRNA level and expressed as a percentage of wild-type
expression. Shown are the mean values, along with standard deviations,
from 3 separate experiments in which 2 samples were used for each
genotype.
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A semiquantitative RT-PCR assay was next used to measure murine
peripheral blood -minor mRNA expression. Peripheral blood RNA was
harvested from homozygous 6I mice
(6I/6I) or wild-type mice
(WT/WT), reverse-transcribed to cDNA, and
analyzed by PCR using a murine -minor primer set or a murine
-actin gene primer set as a control for RNA quality and abundance.
PCR reactions were performed in the presence of
[-32P]dATP and stopped before amplifications reached a
plateau. PCR products were resolved by PAGE, and the radioactivity of
individual bands was quantitated with the Phosphorimager (Molecular
Dynamics). As shown in Figure 2D, the output from the -minor globin
gene was equivalent in 6I/6I and
WT/WT mice.
Globin chain composition in the red blood cells of
6I mutant mice
We next measured the abundance of 6I globin chains
in peripheral RBCs. Figure 3A shows
Triton-acid-urea-PAGE analysis of a globin chain composition of
hemolysates derived from wild-type controls
(WT/WT; lanes 1 and 2), 6I
homozygotes (6I/6I; lanes 3 and 4), and
heterozygotes (WT/6I; lanes 5 and 6). The
levels of WT, 6I, min, and
globin chains were quantitated by densitometry; a mean value was
derived from duplicates run on each gel and expressed as a percentage
of WT. As shown in Figure 3A, 6I globin
exhibited retarded electrophoretic mobility compared with the
WT chain. In the erythrocytes of heterozygous mice,
6I globin represented 40% of all -major globin
chains (Figure 3B). A similar reduction in the abundance of
6I mutant chains was also detected in homozygous mice
relative to that of WT chains in wild-type mice (84%
that of WT). The abundance of -minor (Figure 3B) and
-globin chains in the RBCs of 6I homozygotes,
heterozygotes, and wild-type animals were identical. Therefore, despite
the equal abundance of 6I and wild-type mRNAs, the
mutant -globin chains are less abundant than WT chains in
peripheral red cells, suggesting that a posttranscriptional mechanism(s) contributes to this difference in vivo.
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| Figure 3.
Globin-chain composition in mouse RBCs.
(A) Hemolysates were prepared from the peripheral blood of
6I/6I mice,
w/6I mice, or
WT/WT mice. Globin chains from fresh
hemolysates were resolved on TAU-polyacrylamide gels and visualized
with Coomassie blue. Shown is a representative gel from 4 that
displayed virtually identical protein profiles. Positions of
WT, 6I, min, and globin chains are indicated. (B) The WT,
6I, and min chains were quantitated by
densitometry. An average was calculated from duplicates run on each gel
and expressed as a percentage of wild-type level. Shown are the means,
along with standard deviations, from 4 different gels.
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Expression of the retained PGK-neo cassette in
6I mutant mice
We next examined the expression of the PGK-neo cassette in various
adult 6I/6I mouse organs using an S1
nuclease protection assay (data not shown). Correctly initiated PGK-neo
mRNA was detected at low levels in RNA derived from the hearts, lungs,
kidneys, or livers of these mice, but it was easily detected in RNA
derived from the bone marrow and spleens of
6I/6I mice.
To determine whether the PGK-neo cassette was expressed during
hematopoietic development, we plated bone marrow cells from wild-type
mice or from 6I/6I mice in a
methylcellulose-based culture system. Bone marrow cells were cultured
in the presence or absence of 900 µg/mL (active) G418 (Figure
4). In the absence of G418, there was no
significant difference in the number, size, or morphology of CFU-G-,
CFU-GM-, or BFU-E-derived colonies either from wild-type mice or from
6I/6I mice. The presence of G418 in the
media almost entirely inhibited erythroid and myeloid colony formation
from bone marrow progenitors derived from wild-type mice but had no
effect on the number of colonies derived from
6I/6I mice (Figure 4). Moreover, the
presence of G418 had no effect on the size or morphology of the
hematopoietic colonies derived from the
6I/6I mice (data not shown). These
results suggest that the neomycin phosphotransferase gene in the
-globin gene cluster is active in early erythroid and myeloid
progenitor cells derived from 6I/6I
mutant mice and that the cassette remains active throughout both erythroid and myeloid differentiation.
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| Figure 4.
Colony quantitation.
(A) Quantitation of myeloid colonies (CFU-G and CFU-GM) derived from
the bone marrow cells of either wild type or
6I/6I mice using growth conditions that
permitted myeloid differentiation after 8 days of culture. (B)
Quantitation of erythroid colonies (BFU-E) using conditions that
permitted only erythroid growth after 8 days of culture. There was no
significant difference in the numbers of myeloid or erythroid colonies
derived from either the 6I/6I or the
wt/wt mice in the absence of G418. The
addition of 900 µg/mL active G418 caused near-complete suppression of
colony formation from wild-type bone marrow cells. In contrast, G418
did not suppress myeloid or the erythroid colony numbers (or the sizes
of colonies) derived from the bone marrow cells of
6I/6I mice. Graphs represent means and
standard deviations from 3 separate experiments.
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We next sought to detect neomycin phosphotransferase in individual bone
marrow cells derived from adult 6I/6I
mice. Figure 5A shows a 2-color flow
cytometric analysis of Neo expression in erythroid cells
(Ter-119+), late myeloid cells (Gr-1+), and
CD3+ T lymphocytes derived from the bone marrow cells of
adult WT/WT or
6I/6I mice. Neo-labeling was not detected
in the late myeloid compartment or in T cells. In contrast, a fraction
of 6I/6I Ter-119+
erythroid cells stained positively for Neo (Figure 5A).
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| Figure 5.
Expression of PGK-neo in
6I mice.
(A) Flow cytometric analysis was used to quantitate cells expressing
immunoreactive neomycin phosphotransferase. Bone marrow cells were
first labeled with PE-conjugated monoclonal antibodies specific for
cell surface antigen Ter-119, Gr-1, or CD3. Cells were then fixed in
Cytofix/Cytoperm solution and incubated with rabbit anti-Neo polyclonal
antibody and fluorescein-conjugated antirabbit secondary antibody. Neo
expression is plotted on the x-axis, and surface markers are plotted on
the y-axis. (B) Bone marrow cells from
6I/6I mice were analyzed for the presence
of immunoreactive neomycin phosphotransferase by indirect
immunofluorescence microscopy. Cells were first stained with
PE-conjugated antibody specific for murine erythroid lineage marker
Ter-119, spun onto coverslips, fixed in methanol, and incubated with
rabbit anti-Neo polyclonal antibody and fluorescein-conjugated
antirabbit secondary antibody. Nuclear DNA was visualized with DAPI. A
representative microscopic field is shown for Neo (Neo), Ter-119
(Ter-119), and DNA staining (DNA). A merged micrograph of the Neo and
Ter-119 labeling patterns is also shown (Merge). Arrows indicate,
Ter-119+/Neo+ cells; arrowheads,
Ter-119-/Neo+ cells. Magnification
× 200.
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The expression of the PGK-neo cassette in the erythroid compartment was
confirmed by immunofluorescence analysis, as shown in Figure 5B. As
expected, positive Neo staining was readily detected in a significant
proportion of Ter-119+ erythroid cells (Figure 5B, arrow)
but not in the Gr-1+ or the CD3+ population
(data not shown). We also observed a small percentage of
Neo+ cells in the adult bone marrow that were
Ter-119 (Figure 5B, arrowhead); the lineage of these
Neo+ cells is unknown. However, in view of the results from
the colony assays (Figure 4), these Neo-expressing cells may include
earlier erythroid progenitor cells (BFU-E, CFU-E, or proerythroblasts) that do not express the late erythroid marker Ter-119.
As revealed by immunofluorescence microscopy,
6I/6I mice also contained a small
percentage (less than 2%) of Neo+ RBCs in their peripheral
blood (data not shown). These observations strongly suggest that Neo is
present in reticulocytes, but it has a short half-life in mature
peripheral red cells.
Erythrocyte and hemoglobin characteristics of
6I mice
There was no significant difference between the RBC counts,
hemoglobin concentrations (Hb), hematocrit values (Hct), or other red
cell indices of 6I/6I mice compared with
their wild-type counterparts (Table 1).
The reticulocyte counts of all mutant mice were also normal (3% or less; data not shown). Similarly, heterozygous mice
(WT/6I) also had normal hematologic
values (Table 1) and reticulocyte counts (data not shown).
To determine whether the mutant mice contained irreversibly sickled
RBCs in vivo, fully oxygenated peripheral blood smears were prepared
from wild-type mice and homozygous 6I mice. A
representative microscopic field for each group is shown in the top 2 panels of Figure 6. No irreversibly
sickled cells were observed in the periphery of
6I/6I mice (Figure 6). The same blood
samples were treated in vitro under deoxygenating conditions. The
morphology of erythrocytes from 6I/6I
mice did not change on deoxygenation (Figure 6). Similarly, the mutant
hemoglobin did not precipitate with oxidant stress (data not shown).
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| Figure 6.
Homozygous 6I mice have normal peripheral
red blood cell morphology and normal hematologic parameters.
An oxygenated blood smear was prepared from a
WT/WT mouse or a
6I/6I mouse followed by
May-Grünwald-Giemsa staining, magnification × 100. A sickling
test was performed on the same fresh blood samples by viewing cell
morphology under deoxygenating conditions after incubation in a 1%
solution of sodium metabisulfite under coverslips, magnification
× 200. A representative photomicrograph of each sample is
shown.
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Finally, as shown in Figure 7, the
hemoglobin oxygen-binding curve of hemolysates from
6I homozygous mice was indistinguishable from that of
normal blood, demonstrating that 6I mutant hemoglobin
has a normal P50 value
(WT/WT:8.2 mm Hg;
6I/6I:8.0 mm Hg) and the same Hill
coefficient.
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| Figure 7.
Hemoglobin-oxygen dissociation curves.
Oxygenated
hemolysates from 6I homozygotes
(6I/6I), 6I heterozygotes
(WT/6I), and wild-type mice
(WT/WT), were deoxygenated incrementally
with humidified O2-free nitrogen gas. O2
fractional saturation of a hemolysate was determined by monitoring the
change in absorbance at 555 nm versus O2 partial pressure.
A fitted oxygen dissociation isotherm for each genotype (n = 3 for
all groups) is plotted for derivation of a P50
value.
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Discussion |
In this study, we produced a mutation in the mouse -major
globin gene using homologous recombination in embryonic stem cells. The
mutation, located at the 6 position, changed the Ala residue to Ile,
which allowed us to track the mutation at the level of DNA, RNA, and
protein. The mutant locus also contained a retained PGK-neo cassette
between the -major and -minor globin genes. PGK-neo expression
was detected in the maturing erythroid cells from the bone marrow and
in the RBCs from the peripheries of the mutant mice. The mutant
-major globin gene had an output that was identical to its wild-type
counterpart in the bone marrow, and similarly the output of the
-minor globin gene was also normal. Analysis of 6I
protein abundance revealed that red blood cells derived from heterozygous mice had approximately 40% 6I and 60%
WT chains, suggesting that posttranscriptional events
contribute to the final abundance of the mutant globin chain in
RBCs. Homozygous mice bearing the mutation had normal blood counts,
reticulocyte counts, and peripheral smears and had no evidence for a
sickling phenotype.
Based on previous experiments performed in several laboratories, we
expected the retained PGK-neo cassette to down-regulate the expression
of the -minor globin gene because the PGK-neo cassette is interposed
between the 5' LCR and the -minor gene. Groudine et
al16,17 first demonstrated that selectable marker cassettes interposed between the 5' LCR and the -globin cluster could down-regulate expression of the linked -globin gene. Smithies et al33,34 also showed that mice homozygous for an
insertional disruption of the -major gene with a Neo-resistance
cassette are more anemic than mice homozygous for a naturally occurring deletion of the same gene. This discrepancy in the severity of the
phenotypes in these 2 mutations was thought to result from a
"knock-down" of the -minor globin gene expression caused by the
retained selectable marker gene. In a large series of subsequent experiments, it became clear that retained PGK-neo or PGK-hygro cassettes near the positions of HS1, HS2, and HS3 could reduce expression of the embryonic and the adult globin
genes.18,19,24 These results suggested that if a
selectable marker cassette was retained between the functional LCR and
the target gene with which it interacts, expression of the target gene
could be reduced. Other examples of this phenomenon have been
reported.20-23 For example, a retained PGK-neo cassette in
the granzyme B gene down-regulates expression of a number of granzyme
genes located downstream from granzyme B, but it does not disrupt
expression of the cathepsin G gene located at the 3' end of the
granzyme cluster.21 Similarly, a retained PGK-neo cassette
in the cathepsin G gene down-regulated the expression of a chymase gene
downstream from it but did not affect expression of the granzyme genes
located upstream.21 These results suggest that the
neighborhood effect can be unidirectional, but the "rules" for
neighborhood effects have yet to be established.
We do not yet understand why the -minor globin gene is not
down-regulated by the retained PGK-neo cassette. Our results
demonstrate that the lack of a neighborhood effect is not due to
silencing of the PGK-neo gene in the maturing red cells. We observed
high-level PGK-neo expression in the erythroid compartment, indicating
that "capture" of PGK-neo expression by the LCR does not
necessarily disrupt the interaction between the LCR and the neighboring
globin genes. A simplified LCR competition model, therefore, does not explain our data. One alternative possibility is that because the
-minor gene is located at the furthest distance from the 5' LCR, the
LCR effect may be diminished. It is also possible that 3' HS-1, located
downstream from the -globin gene cluster and closest to the
-minor gene, may be able to compensate for functions provided by the
upstream LCR elements. Finally, it is possible that there is simply
some unique topological chromatin constraint for the neighborhood
effect to occur but that this constraint is not operative with this
particular mutation. Regardless, retention of the PGK-neo cassette
between the -major and the -minor globin genes in this position
has no measurable effect on the output of either gene.
The -globin mutation engineered in this study changed an alanine to
an isoleucine residue at position 6 of the -major globin gene. Amino
acids in the 6 position of the mouse and human genes are different
(Ala vs Glu), as are several surrounding residues (Figure 1). The |
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Abstract
Introduction