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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 719-726
PHAGOCYTES
Insertion of enhanced green fluorescent protein into the lysozyme
gene creates mice with green fluorescent granulocytes and macrophages
Nicole Faust,
Florencio Varas,
Louise M. Kelly,
Susanne Heck, and
Thomas Graf
From the Albert Einstein College of Medicine, Bronx, NY; Artemis
Pharmaceuticals, Cologne, Germany; the European Molecular Biology
Laboratory, Heidelberg, Germany; and the Department of Molecular and
Cellular Biology, Ciemat, Madrid, Spain.
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Abstract |
Pluripotent hematopoietic stem cells have been
studied extensively, but the events that occur during their
differentiation remain largely uncharted. To develop a system that
allows the differentiation of cultured multipotent progenitors by
time-lapse fluorescence microscopy, myelomonocytic cells were labeled
with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene
into the murine lysozyme M (lys) locus and using a targeting
vector, which contains a neomycin resistant (neo) gene flanked
by LoxP sites and "splinked" ends, to increase the frequency of
homologous recombination. Analysis of the blood and bone marrow of the
lys-EGFP mice revealed that most myelomonocytic cells,
especially mature neutrophil granulocytes, were fluorescence-positive,
while cells from other lineages were not. Removal of the neo
gene through breeding of the mice with the Cre-deleter strain
led to an increased fluorescence intensity. Mice with an inactivation
of both copies of the lys gene developed normally and were fertile.
(Blood. 2000;96:719-726)
© 2000 by The American Society of Hematology.
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Introduction |
During blood cell formation hematopoietic progenitors
proliferate and differentiate, ultimately generating 8 well-defined lineages. While some bipotent intermediates in adult bone marrow have
been defined by in vitro colony assays, the existence of other bi-,
tri-, and quatri-potent progenitors remains contentious. The exact
relationships between several hematopoietic lineages remain unclear
because it has not yet been possible to directly observe individual
steps of lineage specification and cell maturation during the division
of mammalian hematopoietic cells. Such studies with developing
nematodes have given invaluable information about lineage
relationships, intermediate progenitors, and programmed cell death
during embryogenesis.1 Time-lapse studies with
colony-forming hematopoietic cells are difficult to perform because
lineage assignments of live cells can only be performed during the late
stages of maturation, when their morphologic features are clearly
recognizable. And, although earlier stages can be recognized by
immunostaining of lineage-specific cell surface markers, this procedure
interrupts colony development. For this reason we have decided to
generate mouse lines in which blood cells from a specific lineage are
labeled in vivo, by expression of different fluorescent proteins. When placed in culture, multipotent progenitors from these mice should become fluorescence-positive as they differentiate into specific cell
types, a process that can be followed by video microscopy.
We generated a mouse line in which EGFP was expressed
specifically in the myelomonocytic lineage. This was accomplished by using homologous recombination to insert the EGFP gene into the lysozyme M ( lys) locus. This latter marker was used because it is expressed specifically in myelomonoytic cells (macrophages and
neutrophil granulocytes)2,3 and is likely to encode a gene
product that is not essential for viability of the animals. Indeed, the
lys knock-in mouse line and the cultures derived from it showed
specific fluorescence in macrophages and neutrophil granulocytes. These
mice should become useful for the analysis of lineage relationships and
for functional studies of myelomonocytic cells in
transplantation experiments employing normal and leukemic mouse models.
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Materials and methods |
A genomic clone of the lys gene,2 covering the
region from  6900 to +9150 relative to the transcriptional start
site,2,4 was isolated from a 129/Sv mouse
genomic library (lPS5) (gift from T. Boehm, Max Planck
Institute, Freiburg, Germany). To generate the targeting construct, a
6.7-kb BamHI/HindIII fragment (position 2400 to +4300) was
subcloned, simultaneously introducing a unique XbaI site at position +9
(20-bp [base pair] 5' of the translation start) and
deleting a 350-bp PvuII/NcoI fragment containing the coding part of
exon 1 (including the start codon) and parts of intron 1. The NotI
site of pEGFP-1 (Clontech Laboratories, Heidelberg, Germany) was
inactivated, and a 1.3-kb fragment containing the thymidine
kinase-neomycin resistant (Tk-neo) cassette from pPNT,6 flanked by LoxP sites, was inserted into the DraIII site of this plasmid. The resulting EGFP-Lox-Tk-neo Lox cassette was cloned into the
XbaI site of the modifiedlys subclone. To flank the targeting construct by Tk genes, it was inserted into the SalI site of
the pKO vector.5 The targeting construct was linearized by
NotI, and while half of the preparation was left untreated as a
control, the other half was ligated to a NotI compatible
splinker.7 The splinker was generated by self-annealing of
the following oligonucleotide:
5'-GGCCGGGTACCGCTTTTGCGGTACCC-3'. The gel-purified fragments were then electroporated into R1 ES cells,8 and
the clones were isolated after positive-negative selection with G418 and ganciclovir.9 The gene targeting event was identified
by Southern blot analysis (Figure 1).
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| Fig 1.
Strategy for insertion of the EGFP gene into the
lys locus.
(A) A schematic representation of the linearized targeting construct
and the genomic locus before and after recombination. The 4 exons of
the lys gene and the probe used are shown as solid black boxes.
The transcriptional direction of lysozyme is indicated by an arrow, the
EGFP gene as a shaded box, and the LoxP-sites as boxes
containing "less than" signs. The Tk-neo cassette, with
its transcriptional direction (arrow), is indicated with an open box,
and the HSV-Tk genes, with their transcriptional directions
(arrows), are indicated with hatched boxes. After linearization, the
targeting construct was either left untreated, nonsplinked (B), or
splinked (C) by ligating to oligonucleotides forming hairpin
"splinkers" in order to protect the fragment ends from
exonucleolytic degradation. The fragments were then electroporated into
R1 ES cells, the cells were cultured in G418 and ganciclovir, and
double-resistant clones were isolated. Finally genomic DNA was
prepared, digested with EcoRI, and analyzed by Southern blot analysis
with the indicated probe. The 9-kb band in panels B and C corresponds
to the fragment obtained from wild type clones; the 6-kb band
corresponds to the fragment generated by homologous recombination in
some clones. (These are indicated with a star).
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Genotypes of lys-EGFP-ki mice were determined by polymerase
chain reaction (PCR) using the following primers:
MLYSUP, 5'-AAGCTGTTGGGAAAGGAGGG-3'; EGFPDWN, 5'-GTCGCCGATGGGGGTGTTCT-3'; and
MLP1, 5'-TCGGCCAGGCTGACT CCATA-3'. The reactions
were performed with 100 ng tail DNA and all 3 primers (0.2 µmol/L
MLYSUP, 0.1 µmol/L EGFPDWN, and 0.1µmol/L MLP1) for 30 cycles each at 30 seconds denaturation at
94°C, 30-second annealing at 60°C, and 60-second elongation at
72°C. The products were analyzed on a 1.5% agarose gel. Primers
MLYSUP and MLP1 amplify a 220-bp product from the wt
allele, whereas MLYSUP and EGFPDWN together amplify a
680-bp product from the knock-in allele.
Blood was collected from tail veins using heparinized capillaries that
were subsequently flushed with phosphate-buffered saline (PBS). The
cells were then treated with lysis buffer (155 mmol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L EDTA [ethylenediamine tetraacetic acid]) to lyse mature
erythroid cells. Cells for cytometric analyses were stained with
ethidium bromide, and dead cells were gated out. To prepare the
microscopic images, an inverted microscope with an oil immersion phase
3 objective (× 60; Olympus, Melville, NY) and an
air-cooled CCD camera (Roper Scientifics, Tucson, AZ) was used. The fluorescent image was deconvoluted using
Hazebuster software (Vaytek, Fairfield, IA) and
Photoshop 3.0 software (Adobe Systems, San Jose, CA). For bone marrow
preparations, mice were killed by cervical dislocation, and the bone
marrow cells were flushed from femur cavities with PBS.
For antibody staining, 105 nucleated cells were pelleted by
centrifugation, washed once in PBS containing 1% bovine serum albumin (BSA), and then resuspended in 10 µL appropriately diluted primary antibody. After 15 minutes of incubation on ice, 150 µL PBS/BSA was
added, and the cells were pelleted, washed once with PBS/BSA, and
resuspended in 10 µL secondary antibody (phycoerythrin-conjugated [PE-conjugated] goat antirat antibody) (Dianova,
Hamburg, Germany). After 10 minutes of incubation on ice, 150 µL
PBS/BSA was added, the cells were centrifuged again, washed once with
PBS/BSA, and resuspended in 150 µL PBS/BSA. Subsequently, the cells
were analyzed by 2-color flow cytometry using a fluorescence activated
cell sorter (FACS) (FACScan, Becton Dickinson, San Jose, CA). Primary antibodies were directed against the following antigens:
Mac-1,10 ER-MP12,11,12 ER-MP20,11
Ly-6G,13 CD3, Ter119, B220, and Sca-1.14 Bone
marrow cells used for sorting were pretreated with lysis buffer.
Sorting was performed with a FACStar Plus (Becton Dickinson), excluding
nonviable cells after propidium iodide staining.
For colony assays in plasma clots, 4 × 104 freshly
prepared bone marrow cells were resuspended in 1 mL IMDM (Iscove's
modified Dulbecco's medium) supplemented with 10% fetal calf serum
(FCS), 0.3 mmol/L monothioglycerol, 50 µg/mL Vitamin C, 200 µg/mL
transferrin, and 5% conditioned medium from L929 cells as a source of
macrophage-colony-stimulating factor (M-CSF). We then added 60 µL
citrated bovine plasma (Sigma Chemical Co, Poole,
England) and 0.5 units of thrombin, and the mixture was
quickly transferred to a 24-well plate, where it was allowed to clot.
After incubation for 6 days at 37°C and 5% carbon dioxide
(CO2) in a humidified incubator, the clots were transferred to slides and dried. Fluorescence micrographs were taken from some of
the colonies before the clots were completely dried, and the colonies
were marked. The cells were then fixed in methanol, stained with a
May-Grünwald Giemsa stain (Diff-Quik; DADE Behring, Dudingen,
Germany), and the marked colonies were photographed under brightfield.
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Results |
To generate mice expressing EGFP, specifically in cells of
the myelomonocytic lineage, the gene targeting vector shown in Figure 1
was constructed. The EGFP gene was inserted immediately downstream of the transcriptional start site of the lys
gene.2 The targeting vector contained 2 copies of the
herpes simplex virus (HSV) Tk gene at the 5' and 3'
ends as a negative selectable marker,9 and the neo
gene was used a positive selection marker. The neo gene was
flanked by LoxP sites, which are recognized by the bacteriophage P1 Cre
recombinase. Homologous recombination will result in loss of the
Tk sequences. Therefore, stable transfectants that have
recombined randomly and retain the Tk gene(s) should be
susceptible to killing by ganciclovir.
In an attempt to enhance the efficiency of homologous recombination, we
tested the effect of the "splinker ligation" technique. This
method is based on the use of hairpin structure-forming
oligonucleotides that protect the transfected construct from
exonuclease attack. It has been shown to improve the efficiency of
negative selection by ganciclovir in murine erythroleukemia cells
transfected with targeting constructs containing the
HSV Tk gene.7 To determine whether the technique can also
be used to increase the relative frequency of homologous recombinants
among G418/ganciclovir double-resistant ES cell colonies transfected
with the targeting construct, 2 parallel experiments were
performed. In the first, the NotI-linearized lysozyme targeting
construct wastransfected without further treatment ("nonsplinked"
construct). In the second, the construct was ligated to NotI-compatible
splinkers, which are designed to protect the (open) DNA ends by hairpin
structures ("splinked" construct). After transfection and
selection with G418 or G418 plus ganciclovir, the colonies were
counted. Although the number of G418-resistant clones was slightly
lower with the splinked targeting construct, negative selection by
ganciclovir was about 3 times more efficient in colonies transfected
with this construct than with the nonsplinked construct (Table
1).
To determine whether the proportion of clones with homologous
recombination is enriched in the double-resistant ES cell colonies transfected with the splinked construct, we made a Southern blot analysis with an external probe. The targeting frequency of the nonsplinked construct was 8%, while the frequency for the splinked construct was 28% (Figure 1 and Table 1), which exactly reflects the
factor of enrichment observed after negative selection. Five clones
that had scored positively in the first round were subjected to
additional Southern blot analyses with different restriction enzymes
and a neo probe. This was done to rule out additional random
integrations and to confirm the correct structure of the targeted locus
(data not shown). After blastocyst injection, 2 of these ES cell clones
resulted in germ line transmission, and the corresponding mice were
designated lys-EGFP-ki.
To analyze EGFP expression in lys-EGFP-ki-positive or
lys-EGFP-ki-negative mice, peripheral blood was collected from
the tail veins of 1 litter of mice at weaning. Peripheral blood
leukocytes were analyzed by flow cytometry, and the mice were genotyped
by PCR. Between 14% and 44% fluorescent cells could be detected in the peripheral blood leukocytes of heterozygous lys-EGFP-ki
mice, whereas no fluorescent-positive cells were seen in their wild type littermates (less than 0.1%) (Figure
2A,B). The PCR bands identified the
genotypes of knock-in mice (Figure 2E). Most fluorescence-positive cells were larger than erythrocytes. They could be identified by their
morphology under epifluorescent illumination as polymorphonuclear granulocytes because they exhibited nuclear structures characteristic for this cell type (Figure 2F). In addition, about 1 in 50 of the green
cells showed band-shaped nuclei characteristic of monocytes. Cells
isolated from the peritoneum contained 5%-15% EGFP+
cells. This percentage increased to about 60% when the cells were
seeded in tissue culture plates for 1 day. Nonadherent cells were
removed, thereby revealing cells with the morphology typical of
peritoneal macrophages. Approximately 10% of positive cells were
extremely bright (Figure 3A). The
proportion of EGFP+ cells did not further increase after a
48-hour incubation with 500 ng/mL lipopolisaccharide (F.V. and T.G.,
unpublished data, July, 1999). Cryosections revealed
the presence of EGFP+ cells in the lung and liver and in
cell suspensions from uteri of mice in estrus (F.V., T.G., and Jian Li,
unpublished data, July, 1999), thereby reflecting the
presence of resident macrophages in these organs.
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| Fig 2.
Analysis of blood leukocytes from lys-EGFP-ki
mice.
(A-D) FACS analysis of blood samples collected from
the tail veins. Red cells were lysed to obtain the leukocyte fraction.
After suspension in PBS, the samples were analyzed for GFP
fluorescence, and the data were plotted as relative fluorescence
intensity (Y axis) versus the relative cell number (X axis).
The numbers in each panel represent the percentage of
fluorescent-positive cells in the respective sample. The following
animals were used: (A) control mice; (B) heterozygous lys-EGFP mice,
not neo-deleted; (C) heterozygous lys-EGFP mice, neo-deleted; (D)
homozygous lys-EGFP mice, neo-deleted. The following
average fluorescence intensities plus or minus SD were calculated
from 4-5 animals in each group: (A) less than 2, (B) 92.6 ± 51.5,
(C) 194.6 ± 50.1, and (D) 625 ± 396.3. (E) Genotypic
analysis. Genomic DNA was prepared from mouse tails and analyzed by PCR
using primers that amplify a 220-bp fragment from the wild type allele
and a 680-bp fragment from the targeted allele. (F) Micrographs of a
fresh blood sample from a lys-EGFP+/ki
mouse were made using phase contrast (left) and fluorescence microscopy
(middle). The picture shown on the right was obtained by overlaying the
2 images. (Scale bar, 10 µm.)
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| Fig 3.
Micrographs of cultured peritoneal macrophages and bone
marrow-derived colonies.
Left image, bright field; right image, fluorescence. (A) Peritoneal
blood macrophages from heterozygous lys-EGFP not neo-deleted (+/ki) and
neo-deleed (+/ki=neo D) mice 1 day after seeding in culture. (B)
Macrophage colonies. Bone marrow cells prepared from a wild type (+/+)
and from a lys-EGFP+/ki mouse were seeded
in plasma clot cultures containing M-CSF. After 7 days, the plasma
clots were partially dehydrated, and fluorescence pictures were taken
of the colonies (right panels). Subsequently, the clots were fixed in
methanol and stained with Diff-Quik, and the identical colonies were
photographed under bright field (left panels).
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To study the EGFP expression in colonies developed in culture,
bone marrow cells of lys-EGFP-ki mice were seeded in plasma clot cultures containing M-CSF. The resulting macrophage colonies contained bright green fluorescing cells that were not seen in similar
colonies obtained from a control wild type mouse (Figure 3B). However,
only about half of the adherent macrophage colonies from the knock-in
mice were fluorescence-positive, with some variegation in fluorescence intensity.
To confirm that the fluorescence is confined to cells of the
myelomonocytic lineage, bone marrow of heterozygous lys-EGFP-ki mice was observed by phase contrast and fluorescence microscopy. Approximately 20% of the cells were fluorescence-positive, of which
about 20% were brightly fluorescent and resembled myelocytes and
neutrophil granulocytes, with occasional monocyte-like cells. The
remaining 80% of the cells were weakly positive, showed mostly cytoplasmic fluorescence, and had a blast-like morphology with larger
nuclei. In addition, we analyzed bone marrow cells of the same mice by
staining them with lineage-specific antibodies (detected by PE-coupled
secondary antibodies), and flow cytometry demonstrated that the
EGFP+ cells expressed the myelomonocytic-specific antigen
Mac-1, the granulocyte-specific Ly6-G (or Gr-1) antigen, as well as the
ER-MP20 antigen, which is specific for macrophage precursors from the monoblast stage onwards (Figure
4).11 In contrast, the cells did not significantly express the ER-MP12 antigen, which is present on
more immature progenitors but is no longer found on
monoblasts.11 Likewise, the GFP+ cells were
largely negative for Sca-1, a marker for early hematopoietic progenitors. Finally, they neither expressed the B-cell specific marker
B220 nor the erythroid-specific marker Ter119, although in both cases a
small proportion of the population scored double-positive.
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| Fig 4.
Immunofluorescence analysis of bone marrow cells.
(A) Bone marrow was prepared from wild type mice
(+/+), lys-EGFP+/ki mice, and mice in
which the neo gene had been deleted
(+/ki-neoD). The cells were stained with various
primary antibodies (indicated on the left), and a PE-labeled secondary
antibody was followed by flow-cytometry analysis. The profiles show GFP
fluorescence (green) on the horizontal axis and antibody-mediated
fluorescence (red) on the vertical axis. The percentage of cells in
each of the 3 quadrants containing fluorescence-positive cells
(antibody only, antibody/GFP, and GFP only) is indicated either within
the respective quadrant or immediately adjacent to it. Specificity of
the antibodies: CD3, T cells; Mac-1, macrophages and myelomonocytic
cells; ER-MP12, immature monocytic cells; ER-MP20, monocytic (and some
granulocytic) cells; Ly6-G (Gr-1), neutrophil granulocytes; Sca-1,
early multilineage progenitors; Ter119, erythroid cells; and B220,
cells of the B-cell lineage. (B) Micrograph of live cells from a
lys-EGFP-ki/ki-neoD mouse. Cells were stained with
anti-B220 antibody coupled to PE and photographed under a brightfield
to reveal antibody-positive cells (red surface staining) as well as
under epifluorescence illumination to reveal EGFP+ cells
(green cells). The field contains 9 B220+ cells, 11 EGFP+ cells, and 2 double-negative cells
(as well as 2 cell ghosts).
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Figure 4 also shows that a small percentage of ER-MP20+
cells scored negative for EGFP fluorescence. This could be due to the fact that EGFP is only expressed by the most mature
myelomonocytic cells or because levels of EGFP were below
detection. Alternatively, it might be due to an interference of the neo
selection marker with expression of the targeted gene, as had been
demonstrated for a similar knock-in of the  globin
locus.15 We therefore deleted the floxed neo gene
by crossing lys-EGFP-ki mice with a Cre-deleter
strain.16 Heterozygous animals of both strains were
crossed, and the offspring was analyzed by Southern blot analysis. Out
of 18 animals, 10 had inherited the lys-EGFP-ki allele. Of
these, 6 animals showed 100% deletion of the neo cassette in
their tail DNA, whereas 4 animals showed no detectable deletion (data
not shown). It is thus highly likely that the neo cassette was deleted
in all mice that had inherited the lys-EGFP-ki allele as well
as the Cre transgene. Animals that showed deletion of the
neo gene in their tail DNA transmitted only the
neo-deleted form to their progeny, indicating that the deletion
had also occurred with 100% efficiency in germ cells.
There was a slight increase in the number of EGFP+ cells in
the blood leukocytes of heterozygous Lys EGFP mice (Figure 2C). However, there was a statistically significant (2-fold) increase in
fluorescence intensity, which increased another 3-fold in the neo-deleted homozygous lys EGFP knock-in animals
(Figure 2, legend). The proportion of EGFP+ peritoneal
macrophages also increased in the cells derived from neo-deleted animals (from about 60% to 95%) (Figure 3B),
although the very bright cells seen before deletion were no longer
apparent. Also, the proportion of EGFP+
myeloid type colonies obtained from bone marrow increased
significantly. The overall expression level of EGFP in the bone
marrow increased somewhat again, with approximately a 2-fold increase
in the EGFP fluorescence intensity of ER-MP20+ and
Ly6-G+ cells in neo-deleted mice (Figure 4, right
column). We therefore conclude that expression of the knocked-in
EGFP gene was impaired by the neo cassette.
To further characterize the identity of the EGFP-expressing
cells, bone marrow cells were sorted by FACS into GFP-high,
GFP-low, and GFP populations (Figure
5A,B). They were then cytocentrifuged onto slides, stained with benzidine and Diff-Quik, and evaluated by microscopic inspection (Figure 5C). The highly GFP+
fraction contained 52% mature neutrophil granulocytes, 40% myelocytes (promyelocytes and metamyelocytes), 4% monocytes, and 3% nonmyeloid cells. The weakly GFP+ cells contained 13% granulocytes,
80% myelocytes, and 4% other cells. Finally, the
GFP cells comprised 85% erythroid and lymphoid
cells and only 4.5% myelomonocytic cells.
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| Fig 5.
Morphology of EGFP+ cells sorted from the
bone marrow of lys-EGFP+/ki mice.
Bone marrow cells were prepared and sorted by FACS. (A) FACS profile
showing relative fluorescence intensity of GFP+ cells
versus forward scatter, a parameter that is proportional to the cell's
volume. The profile was subdivided by gating into a highly positive
fraction (2 × 103 to 1 × 104,
top quadrant), a moderately positive fraction
(4.5 × 101 to 2.3 × 102, middle
quadrant), and a negative fraction (100 to 101,
bottom quadrant). (B) Cell profiles are shown from these 3 gated areas
after sorting. (C) Micrographs of the sorted cells stained with
Diff-Quik (a May-Grünwald Giemsa-like stain).
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Discussion |
Hematopoietic cells from the myelomonocytic lineage can be labeled
in vivo by inserting the EGFP gene into the lys locus. Perhaps surprisingly, the most brightly positive cells were found to be
mature neutrophil granulocytes, followed by their precursors and by
monocytic cells. This mouse line should enable us to visualize the
transition of multipotent progenitors to myelomonocytic cells in
culture by observing the onset of EGFP expression using
time-lapse fluorescence microscopy.
The observed 3-fold increase in the proportion of homologous
recombinant ES cell clones, through the use of a splinker-protected targeting construct, indicates that this technique will be useful for
introducing other targeting vectors that contain the Tk gene as
a negative selection marker. This should also make it easier to
generate mice expressing different GFP forms in various hematopoietic lineages because the number of clones to be analyzed can be
significantly reduced, and thus several transfections can be performed
in parallel. It has been suggested that the splinkers act by protecting
the transfected DNA fragment from exonucleolytic degradation before integration, thus preventing destruction of the Tk gene. If the gene is destroyed, ganciclovir-resistant colonies will exhibit random
integrations.7 A positive effect of the splinkers on the
general transfection efficiency would be expected if the neo gene, which is located more centrally in the construct, would be
affected by exonuclease attack. However, this was not the case in our
experiments; the total number of neo-resistant colonies was not
elevated in the cultures transfected with the splinked construct
relative to the nonsplinked control.
Following the cross of lys-EGFP-ki mice with a
Cre-deleter strain, deletion of the neo cassette from
the integrated targeting construct resulted in a significant increase
in fluorescence intensity without altering the specificity. This has
also been observed for the globin locus flanked by a floxed
neo cassette.15 However, the effect seems to be
more complex because cell populations from the original mice contain a
fraction of extremely bright fluorescent cells that are no longer seen
in the neo-deleted populations (compare Figure
2B,C). The molecular mechanism by which the neo cassette modulates EGFP expression remains to be determined.
As expected from the expression of the lys
gene,2,17 the inserted EGFP gene was specifically
activated in myelomonocytic cells. These data are also in agreement
with a recently described knock-in of the Cre gene into the
lys locus, which led to Cre activity in macrophages and
granulocytes.18 For reasons that are unclear, the
proportion of EGFP+ cells in the peripheral
blood of the lys-EGFP knock-in mice showed great variability
between animals, while there was less variability between bone marrow
preparations. One possibility is that these variations reflect
differences in exposure to bacterial pathogens.2,19 Indeed, the 2 animals with the highest proportion of myeloid
cells observed (the last 2 animals shown in Figure 2D) were males that exhibited wounds incurred through fighting.
A small proportion of erythroid cells (defined by expression of the
Ter119 marker) and B cells (expressing the B220 marker) were also
EGFP+. Reconstruction experiments with
lysates from EGFP-expressing cells incubated with normal
erythrocytes (F.V. and T.G., unpublished data, May,
1999) ruled out the possibility that the double-positives represent
erythroid cells that had nonspecifically bound EGFP. Another
possibility is that they correspond to myelomonocytic cells that
have ingested erythroid cells.20 However, the most likely
explanation is that they represent artifacts of aggregation because the
proportion of double-positives was dramatically reduced when the blood
samples were diluted before FACS analysis.
The targeting vector was constructed in such a way that the lys
gene of the targeted locus is no longer transcribed or translated. Therefore, mice homozygous for the knock-in vector should not make a
functional lysozyme protein. Nine homozygous animals (4 containing the
neo gene and 5 without the gene) grew to normal sizes, they
could be bred as homozygous strains, and all contained macrophages.
However, a more detailed examination of specific macrophage and
neutrophil functions (such as phagocytic capacity and bactericidal
action) of the lys-defective animals remains to be performed.
The lys-EGFP-ki mice will be useful in
reconstitution/transplantation experiments of myelomonocytic cells
because small numbers of donor-derived fluorescence-positive cells can
be easily identified in recipient animals. If the mice are crossed with
appropriate myeloid leukemia models, the
EGFP+ cells might help to identify leukemic
cells and to monitor their ablation during treatments aimed at curing
disease. They might also be useful in monitoring granulocyte
infiltration as a response to bacterial infections. Most importantly,
however, the lys-EGFP-ki mouse model will enable us to perform
time-lapse fluorescence microscopy of developing colonies in vitro,
under conditions that avoid interfering with the spatial arrangements
of cells within the colony. This technique could be combined with the
staining of hemoglobin-expressing cells in the colonies, thereby
revealing cells of erythroid origin (A. Sivunen, F.V., and T.G.,
unpublished data, July, 1999). Then it would be
possible to reconstruct the path by which these cells are formed from
the colony founder, to clarify whether hematopoiesis proceeds in a
hierarchical manner, and to determine the influence of cytokines on
lineage commitment. The answers to some of these questions should not
only improve our general understanding of the differentiation and
function of myelomonocytic cells, but they might ultimately lead to
practical applications such as gene therapy approaches.
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Acknowledgments |
We thank Kelly McNagny for help with flow cytometry and
cytospins, Jonathon Homeister for the Ly-6G antibody, Karen
Brennan for blastocyst injections, Ruediger Klein and Richard
Stanley's group for reagents and discussions, and Michael Cammer,
Analytical Imaging Facility of AECOM, for help with the imaging.
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Footnotes |
Submitted September 7, 1999; accepted March 6, 2000.
F.V. is a recipient of the Beca de
Perfeccionamiento de Doctores of the Ministerio de Educación y
Cultura de España, Spain. S.H. was sponsored by a postdoctoral
fellowship of the DAAD, Bonn, Germany.
Reprints: Thomas Graf, Albert Einstein College of Medicine,
1300 Morris Park Ave, Chanin 302, Bronx, NY 10461; e-mail: graf{at}aecom.yu.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction