|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1855-1863
Promoter Elements of vav Drive Transgene Expression In Vivo
Throughout the Hematopoietic Compartment
By
Sarah Ogilvy,
Donald Metcalf,
Leonie Gibson,
Mary L. Bath,
Alan W. Harris, and
Jerry M. Adams
From The Walter and Eliza Hall Institute of Medical Research, Royal
Melbourne Hospital, Victoria, Australia.
 |
ABSTRACT |
To develop a method for targeting expression of genes to the full
hematopoietic system, we have used transgenic mice to explore the
transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we
showed that a cluster of elements surrounding its promoter could drive
hematopoietic-specific expression of a bacterial lacZ reporter
gene, but the expression was confined to lymphocytes and was
sporadically silenced. Those limitations are ascribed here to the
prokaryotic reporter gene. With a human CD4 (hCD4) cell surface
reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types.
In multiple lines, hCD4 appeared on most, if not all, B and T
lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and
nucleated erythroid cells. Moreover, high levels appeared on both
lineage-committed progenitors and the more primitive preprogenitors. In
the fetus, expression was evident in erythroid cells of the definitive
but not the primitive type. These results indicate that a prokaryotic
sequence can inactivate a transcription unit and that the vav
promoter region constitutes a potent transgenic vector for the
entire definitive hematopoietic compartment.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ALTHOUGH MUCH HAS BEEN learned about the
regulation of genes expressed in specific hematopoietic
lineages,1 little is known about multilineage regulation.
Hence, the vav gene is of particular interest. vav is
expressed in virtually all hematopoietic cell lines and, commencing in
the fetal liver, in all normal hematopoietic cell types but in very few
others.2-7 As predicted from its sequence,3 Vav
transduces signals from various surface receptors to Rho-like G
proteins.8 Although vav is not, as was first
reported,7 required for mouse development,9 it
is essential for full lymphocyte development and
function.9,10 Whether its absence affects nonlymphoid
hematopoietic cells has not been clearly reported.9
Clarifying the transcriptional regulation of vav might allow
expression of any gene to be targeted to the entire hematopoietic system, providing a new avenue for addressing issues such as lineage commitment and the basis of leukemogenesis. We have therefore attempted
to identify vav regulatory elements active in the most stringent test, the transgenic mouse. We first identified sites where
the vav chromatin had been rendered hypersensitive (HS) to
DNase-I, presumably by bound transcription factors.11 All 5 HS sites found, which span a 14-kb region across the first exon (Fig 1A), appeared in different
hematopoietic cell types but not in nonhematopoietic cells and
therefore constituted prime candidates for the key regulatory elements.
Transgenes that included 4 or 5 of these sites and the widely used
Escherichia coli lacZ ( -galactosidase) reporter gene were
active in hematopoietic and not other tissues, but the expression was
invariably confined to lymphocytes and was always variegated, ie,
restricted to a proportion of the cells of a given type.11
Moreover, that proportion decreased in a fashion consistent with
stochastic inactivation.
View larger version (12K):
[in this window]
[in a new window]
| Fig 1.
Relationship of the vav-hCD4 transgenes to the
vav locus. (A) 5' end of the mouse vav locus
showing the 5 hematopoietic-specific HS sites ( ) surrounding exon 1 ( ), as well as a testis-specific promoter (t) near exon 2 (see
Discussion). (B) Expanded map of the vav regions used in the
transgenes, with the untranslated portion of exon 1 shown unfilled. (C)
The HS321/45 vav-hCD4 transgene. (D) The HS21/45 transgene,
showing 2 PCR primers (a and b) used in its
construction (see Materials and Methods). (E) The mutant hCD4 protein
from which the reporter was derived. ss, splice sites; pA,
polyadenylation region F43I, the mutated extracellular residue; TM,
transmembrane region. Restriction sites are K, Kpn I; R,
EcoRI; B, BamHI; S2, Sac II; N, Nco I;
Ne, Nae I; Hp, Hpa I; H3, HindIII; those in
parentheses have been destroyed, whereas those in bold were used to
excise the transgenes from the plasmid backbone; for more detailed
maps, see Ogilvy et al.11
|
|
As reviewed recently,12,13 variegated expression, first
observed in Drosophila and yeast, has now been recognized in mice with
a number of transgenes. The phenomenon favors the view that the
activity of any transcription unit is primarily determined for each
cell in a binary, all-or-none fashion.14 The probability of
transgene activity in a given cell is believed to be set by competition
between its positive regulatory elements, such as a promoter, enhancer,
or locus control region, and negative influences from surrounding
chromatin.12,13,15
Although the limitations in vav-lacZ expression might have
reflected the need for unidentified vav regulatory elements, it seemed possible that vav regulation had instead been
compromised by the bacterial reporter, which has sometimes distorted
transgene expression (see Discussion). Indeed, we report here that
substitution of a mammalian reporter allowed vav transgene
expression throughout the hematopoietic compartment. These results
strongly implicate the prokaryotic reporter in transgene silencing and
demonstrate that the vav regulatory elements provide a powerful
transgenic vector for the hematopoietic system.
| |
MATERIALS AND METHODS |
Generation of reporter, transgenes, and transgenic mice.
The hCD4 reporter (Fig 1D and E) was derived by polymerase
chain reaction (PCR) with a proof-reading polymerase from a cDNA (the
kind gift of Dr Toshiko Sakihama, Dana-Farber Cancer Institute, Boston,
MA) bearing a mutation (F43I) reported to preclude CD4 association with major histocompatibility complex (MHC)
class II.16 The 5' primer introduced a mammalian
consensus initiation sequence, whereas the 3' primer,
corresponding to nucleotides 1337-1350 of the cDNA
sequence,17 excluded the region encoding the C-terminal 33 amino acids, the domain required for signaling.18 By
overlap PCR, the truncated hCD4 cDNA was placed between an intron from the SR expression vector19 and the SV40 late
region polyadenylation signal (nucleotides 2538-2746 in GenBank J02400; Fig 1D). The cassette was bounded by two 48-bp recognition sequences (FRT) for the Flp recombinase to provide the option (not yet tested) of
using Flp-mediated recombination to aid in replacing the hCD4 reporter
by another gene.
The new transgenic vectors (Fig 1) were derived from HS21/45
vav-gal.11 To eliminate the lacZ
sequences and introduce convenient restriction sites, we used that
vector as a template in an inverted PCR with primers (a and
b in Fig 1D) flanking the lacZ sequence but pointing
away from it. The proof-reading Tth polymerase was used to generate the
9.4-kb PCR product bearing 2.3 kb of vav sequences upstream
from the promoter, the 2.7-kb plasmid backbone, and 4.4 (0.7 + 3.7) kb
of sequences from the vav first intron. Insertion of the
hCD4 cassette yielded HS21/45 vav-hCD4 (Fig 1D). To
construct the vector including HS3 (Fig 1C), a HindIII-digested HS21/45 transgene fragment was blunt-end ligated to a Not
I-digested plasmid containing 5.5 kb of additional upstream sequences
(kindly provided by Dr Bernd Hentsch, Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia).
In these constructions, all PCR steps used high fidelity polymerases,
and the final constructs were extensively mapped with restriction
endonucleases. The CD4 expression cassette was partially sequenced to
confirm the junctions and the presence of the F43I mutation.
For microinjection, each transgene was separated from the vector by
electrophoresis of digests (HindIII for HS21/45
vav-hCD4 or Sac II for HS321/45 vav-hCD4)
through low melting point agarose, purified using an Elutip-d
(Schleicher and Schuell, Dassel, Germany), precipitated with ethanol,
and dissolved in sterile 10 mmol/L Tris-HCl (pH 7.4), 0.1 mmol/L EDTA.
Microinjected (C57BL/6J × SJL/J) F2 eggs were transferred to
pseudo-pregnant (CBA/H × C57BL/6) F1 females.20
Transgenic pups were identified by PCR on DNA from tail biopsies with
primers specific to the transgene SV40 sequence.
Flow cytometric analysis.
Peripheral blood leukocytes, obtained after NH4Cl lysis of
erythrocytes, were incubated on ice with phycoerythrin (PE)-labeled mouse antihuman CD4 monoclonal antibody (MoAb; RPA-T4; Pharmingen, San
Diego, CA) plus anti-Fc receptor antibody (2.4G2) to reduce nonspecific binding.21 Viable stained cells (>10,000)
were analyzed on a FACScan (Becton Dickinson, San Jose, CA) as
described.22 Single-cell suspensions from femoral bone
marrow, thymus, spleen, and mesenteric lymph nodes were analyzed
similarly, except that mature red blood cells were gated out by forward
scatter rather than lysed. Nontransgenic (C57BL/6J × SJL/J) F1
and littermate mice provided routine controls. For cell lineage
analysis, cells were incubated simultaneously with a fluorescein
isothiocyanate-labeled antibody to a cell surface marker, PE-labeled
anti-hCD4 MoAb, and 2.4G2 and analyzed as above. The MoAbs were against
B220 (RA3-6B2), mouse CD4 (H129.19.6.8), CD8 (YTS-169), Thy1
(T24.31.2), IgM (anti-Cµ.5.1), IgD (11-26C), Mac1 (M1/70), Gr1
(RB6-8C5), and Ter119.11,22
With vav-lacZ mice, -galactosidase activity in individual
blood cells was determined by the fluorescence-activated cell sorting (FACS)-gal assay,23 as detailed
previously.11
To analyze fetal blood or liver, pregnant females were killed, and the
closed uterus was washed. The uterine wall and yolk sac were removed
without disrupting the umbilical cord, and the umbilical vessels were
clamped and severed. Each washed embryo was bled into wash buffer by
severing the jugular veins and cervical arteries. Cytocentrifuge
preparations of fetal blood were stained with DiffQuik (Lab Aids,
Narrabeen, New South Wales, Australia). Cell suspensions from E12.5 and
E14.5 fetal liver were made by passage through a 21-gauge needle. Each
fetus was genotyped by PCR.
FACS sorting and culture of bone marrow cells.
To obtain cells differing in hCD4 level, femoral bone marrow cells from
adult HS21/45 vav-hCD4 mice were treated with PE-anti-hCD4 MoAb, unlabeled anti-Fc receptor antibody, and 1 µg/mL propidium iodide and sorted (MoFlo; Cytomation, Fort Collins, CO) by viability and hCD4 staining (see Fig 4). Cytocentrifuge preparations were stained
with May-Grunwald-Giemsa to enumerate different cell types. For colony
assays (see Table 3), 25,000 nucleated cells were cultured for 7 days
in semisolid medium as described.24 The dried cultures were
stained, first for acetylcholinesterase activity, then with Luxol fast
blue, and finally with hematoxylin, and were scored microscopically to
verify colony counts and to determine the cell composition of each colony.
Immunohistochemistry.
Tissues embedded in OCT (Tissue-tek; Miles, Elkhart, IN) were
snap-frozen, and 5-µm sections were fixed in acetone. Aldehyde groups
were blocked with 0.2 mol/L glycine, endogenous peroxidase quenched
with 3% H2O2, and endogenous biotin and avidin
blocked using reagents from Vector Laboratories (Burlingame, CA). The sections were then incubated 2 hours at room temperature in a 1:20
dilution of biotin-conjugated mouse anti-hCD4 MoAb (as described above), developed using a Vectastain Elite ABC (peroxidase) kit (Vector
Laboratories) with diaminobenzidine as substrate and counterstained with hematoxylin.
| |
RESULTS |
Extensive expression of a CD4 reporter in the blood.
We wished to use a mammalian cell surface reporter to facilitate
cell-by-cell (ie, flow cytometric) analysis and chose human CD4 because
its molecular interactions have been well characterized and MoAbs are
available that recognize it specifically. To preclude interference with
normal physiology, the cDNA used was truncated so that the polypeptide
(Fig 1E) would lose its intracellular signaling capacity,18
whereas a mutation in its extracellular domain (F43I) reportedly
prevents association with MHC class II, the major CD4
ligand.16
The 2 vav-hCD4 transgenes were based on the most effective
vav-lacZ transgenes.11 One (Fig 1D)
contains the 2 proximal upstream HS sites and both intron sites
(hence HS21/45), whereas the other, HS321/45 (Fig 1C), also bears the
distal upstream HS3. Both maintain the normal order of the HS sites,
with the hCD4 cDNA replacing the coding portion of vav exon 1 (Fig 1B and C). Analysis of 64 primary transgenic mice showed that both
transgenes were highly effective. Flow cytometry on their peripheral
blood cells typically showed substantial hCD4+ cells
(Fig 2B through D). HS21/45
vav-hCD4 was expressed in 27 of 35 primary mice (77%),
and the larger construct was expressed in a similar proportion (25 of
29 [86%]). The proportion of animals with low, medium, or high
expression was also similar for the 2 transgenes.
View larger version (32K):
[in this window]
[in a new window]
| Fig 2.
Flow cytometric analysis of transgene expression in
peripheral blood leukocytes. (A) A vav-lacZ transgenic progeny
mouse (10 weeks old) and (B through F) HS21/45 vav-hCD4 mice.
(B) Primary vav-hCD4 animal with intermediate expression; (C
and D) mosaic primary vav-hCD4 mice; and (E and F) their
respective transgenic progeny. Dotted lines with shading underneath
show nontransgenic controls run in parallel. The expression of
lacZ was assessed by a flow cytometric assay for
-galactosidase activity11 and of hCD4 with an antibody
(see Materials and Methods). The 2 peaks in (E), which may reflect the
different hCD4 levels in different cell lineages (see Table 1), were
not as notable in other mice of that strain.
|
|
In contrast to a typical vav-lacZ line (eg, Fig 2A), many
primary animals bearing either vav-hCD4 transgene showed
expression in essentially all peripheral blood leukocytes (eg, Fig 2B).
The hCD4-negative population in the remaining primary mice (eg, Fig 2C
and D) might reflect variegation or simply genetic mosaicism, due to
transgene insertion after the 1-cell stage. In strong support of the
latter interpretation, all the transgenic descendants of 5 primary
animals showing a heterocellular pattern (2 HS21/45 and 3 HS321/45)
exhibited only hCD4+ blood cells (Fig 2E and F). Hence,
unlike the heritable variegation in all vav-lacZ
mice,11 the heterocellularity encountered in some
primary vav-hCD4 animals can be ascribed to genetic mosaicism.
Expression in all major hematopoietic lineages.
In hematopoietic tissues of progeny animals, concomitant flow
cytometric analysis of immunofluorescence for hCD4 and lineage-specific surface markers showed high transgene activity in multiple lineages (Fig 3 and
Table 1). In the bone marrow (Fig 3A
through E), hCD4 appeared on all B-lineage (B220+) cells,
including those at the more mature IgM+ and
IgD+ stages, as well as on all Mac1+
(CD11b+) and all Gr-1+ cells, which include the
monocytes/macrophages and granulocytes. Most if not all nucleated
erythroid (Ter119+) cells were also positive, albeit at a
lower mean level (Fig 3E). Similarly, in the spleen, all T-lymphoid
(Thy1+) cells bore hCD4 (Fig 3F), including both the helper
(mCD4+) and cytotoxic (mCD8+) mature T-cell
subsets (data not shown).
View larger version (47K):
[in this window]
[in a new window]
| Fig 3.
Expression profile of hCD4 in hematopoietic tissues of
HS21/45 vav-hCD4 progeny mice. Nucleated bone marrow (A)
B220+, (B) IgM+, and (C) IgD+
B-lymphoid cells; (D) Mac1+ myeloid cells; (E)
Ter119+ erythroid cells; and splenic (F)
Thy1+ T cells. Quadrants were set so that the
marker+ hCD4+ cells in a nontransgenic
control sample, run in parallel, did not exceed 5% of total
marker+ cells analyzed. As would be expected, essentially
all cells from the nontransgenic controls then fell in the left
quadrants, and the percentage of cells bearing each lineage-specific
marker (eg, B220) was similar to that of the corresponding transgenic
population.
|
|
The 2 vav-hCD4 transgenes produced similar patterns of
expression in the 8 independent transgenic lines analyzed. With both, hCD4 appeared on virtually all nucleated cells from primary
hematopoietic organs (bone marrow and thymus) as well as secondary
tissues such as spleen. In different lines bearing the same transgene
construct, the level varied over a 10-fold range, probably due to both
insertion position and copy number, but the average values for the 2 constructs were similar. Moreover, the relative levels of hCD4 on
different cell types were consistent, as indicated in Table 1, in which estimates of the fluorescence intensity on each cell type have been
normalized to that on T cells. With both the transgene constructs, lymphoid cells displayed a 3- to 5-fold higher level than myeloid, whereas erythroid cells exhibited the lowest (Fig 3 and Table 1). The
level in nucleated erythroid cells averaged approximately 15-fold lower
than in lymphoid cells.
High activity in clonogenic progenitor cells.
Because essentially all the nucleated cells in bone marrow bore hCD4,
we wished to determine whether clonogenic hematopoietic precursor cells
also expressed the transgene. To allow such studies, we first sorted
all the marrow cells into arbitrary fractions having a low-negative,
intermediate, or high level of hCD4 (Fig 4). Cytocentrifuge preparations of these fractions (data not shown) confirmed that every nucleated cell population displayed substantial hCD4. The low-negative fraction was greatly dominated by erythrocytes, and almost the only nucleated cells present were approximately 20% of
the mature granulocytes. The intermediate fraction contained the
majority of both the mature granulocytes and the nucleated erythroid
cells. Most of the lymphocytes, eosinophils, and blast cells were
found in the hCD4hi fraction.
View larger version (22K):
[in this window]
[in a new window]
| Fig 4.
Fractionation of transgenic bone marrow cells by hCD4
level. Flow cytometric analysis of cells from a HS21/45
vav-hCD4 mouse incubated in the absence (A) or presence (B) of
a labeled anti-hCD4 MoAb. Cells within windows a, b, and c were sorted
for clonogenic assays. In the representative experiment shown (1 of 3),
8% of viable nucleated cells fell in fraction a (low to negative for
surface hCD4), 42% in fraction b (intermediate hCD4), and 50% in
fraction c (high hCD4). Most erythrocytes fell in fraction a and the
dead cells (9% of the total cells) in fraction d.
|
|
Table 2 gives the results of colony assays
for progenitor cells in the fractionated populations, using 3 different
stimuli. Control unstained (or stained but unsorted) populations showed that neither staining nor sorting compromised clonogenicity, and the
colony types produced by each stimulus on the sorted progenitors mirrored those from unfractionated marrow. Significantly, regardless of
the stimulus, the hCD4hi fraction yielded more than 92% of
the total progenitors, and virtually none appeared in the low-negative
fraction (Table 2). hCD4 was present on progenitors committed to
produce colonies of granulocytes, granulocytes and macrophages,
macrophages, eosinophils, or megakaryocytes. Similarly, the more
ancestral preprogenitor cells, which yield colonies of blast cells in
response to stem cell factor (SCF) or to interleukin-3 (IL-3) plus
thrombopoietin, all resided in the hCD4hi fraction (Table
2). Because their frequency was the same or higher than from the
unfractionated marrow, selective losses are unlikely. Thus, the
transgene was very active in all the hematopoietic precursor cells
assayed, including very primitive ones.
Expression in fetal definitive but not primitive erythrocytes.
In the embryo, vav expression appears first in the liver, where
it becomes just detectable at day 11.5 (E11.5) and stronger at
E12.5.3,4 At that stage, the liver has begun to replace the
yolk sac as the major hematopoietic organ and is composed very largely
of the definitive (enucleated) erythrocytes produced there.25 At E12.5, transgenic liver cells consistently
showed an hCD4-associated fluorescence above that in the nontransgenic population (Fig 5A),
indicating that hCD4 was present at a low level on most of the cells
and at a higher level on a small subpopulation. E14.5 liver cells gave
a similar result, with a somewhat higher hCD4 signal (data not shown).
The majority of the dominant erythroid (Ter119+)
population, which represented approximately 95% of the fetal liver
cells (Fig 5C), expressed hCD4 weakly, and the
Ter119 cells (~5%) expressed higher levels (Fig
5D). Indeed, substantial hCD4 appeared on essentially all of the
approximately 2% of liver cells bearing Mac-1 (Fig 5B), as well as on
minor populations bearing Thy1, B220, or Gr-1 (data not shown). Thus,
the vav transgene was clearly active during definitive
hematopoiesis in the fetus.
View larger version (26K):
[in this window]
[in a new window]
| Fig 5.
Flow cytometric assay for hCD4 expression in
fetal liver (A through D) and fetal blood (E through J). (A) Viable
liver cells at E12.5 from a vav-hCD4 fetus (unbroken line) or a
nontransgenic littermate (dotted line and shading). (B) Transgenic
E14.5 liver, also analyzed for Mac-1. (C) Nontransgenic and (D)
transgenic cells at E14.5, also analyzed for Ter119. (E, G, and I)
Nontransgenic and (F, H, and J) transgenic blood (unbroken lines),
superimposed on the nontransgenic profiles (dotted lines and shading).
d indicates the presumptive definitive erythroid population and p the
primitive one. In (E), the unidentified second, very small peak (~2%
of cells) probably represents larger, nonerythroid cells. Results in
(A) are representative of analyses on more than 15 of each genotype, (B
through D) on at least 3 transgenic and 5 nontransgenic littermates,
and (E through J) on 15 to 27 mice. All analyses were performed on mice
of 1 HS21/45 and 2 HS321/45 lines. Exceptions were E12.5 fetal liver
and blood, studied in 1 line for each transgene, and cell surface
marker analysis, which was performed on mice of 1 HS21/45 line.
|
|
The vav gene itself may not be active during primitive
erythropoiesis (see Discussion). To determine whether the transgene was
active in those cells, we first examined fetal blood before the
transition to the definitive stage. At E10.5, primitive (nucleated) erythrocytes derived from the yolk sac greatly predominate, as evidenced by their overwhelming abundance in cytocentrifuge
preparations. Importantly, at that stage the major population in the
transgenic blood never gave an hCD4 signal above the nontransgenic
background (compare Fig 5E and F). The approximately 2% of
hCD4+ cells present specifically in the transgenic blood
(Fig 5F) probably represented nonerythroid cells (eg, monocytes) of
yolk sac origin.
Examination of fetal blood during the transition to definitive
hematopoiesis (E12.5 and E14.5) also failed to show transgene expression in the primitive erythrocytes. As expected, cytocentrifuge preparations showed that, by E12.5, definitive (enucleated)
erythrocytes of liver origin had become as abundant as the primitive
nucleated cells. Unexpectedly, FACS analysis of nontransgenic blood
consistently resolved the 2 populations (Fig 5G). We attribute this
separation to the high autofluorescence of the (larger) primitive
erythrocytes, which was also noted recently by Trimborn et
al26; consistent with that conclusion, the peak
denoted p was always much less prominent at E14.5 (Fig 5I), when the
definitive cells dominated the cytocentrifuge preparations. In the
transgenic blood at these stages, comparison with the superimposed
nontransgenic profile (shaded area) indicated that only the presumptive
definitive population (d) showed a weak but highly reproducible hCD4
signal (Fig 5H and J). Thus, no evidence of vav transgene
activity in primitive erythrocytes was found.
No expression apparent in nonhematopoietic organs.
Immunohistochemical analysis of various organs suggested that
vav-hCD4 expression probably is confined to hematopoietic cell types (Fig 6). As expected, no
hCD4-associated immunoperoxidase (brown) staining was evident in the
nontransgenic spleen (Fig 6B), whereas most cells in transgenic spleen
stained well (Fig 6A), with the highest levels in the lymphoid white
pulp and the megakaryocytes (marked M). Similarly, nearly all cells in
the thymus stained strongly, with the level seemingly higher in the cortex than the medulla (data not shown). These results mimic those
reported for vav itself.4 In contrast, hCD4
appeared in only very rare cells in the heart (Fig 6C), perhaps of
hematopoietic origin, and none was detected in testis (Fig 6D) or
various brain sections (Fig 6E) or in liver or kidney, although their
nonspecific background was higher, probably due to endogenous
peroxidase or biotin.
View larger version (62K):
[in this window]
[in a new window]
| Fig 6.
Immunohistochemical detection of hCD4 in organs of
vav-hCD4 and control mice. (A) Transgenic spleen with a
megakaryocyte (M); (B) nontransgenic spleen; (C) transgenic heart; (D)
transgenic testis; and (E) transgenic cerebellum (other brain sections
were also negative). Nontransgenic brain and testis were
indistinguishable from those shown, and nontransgenic heart essentially
so (see text).
|
|
No decrease in vav-hCD4 expression with age.
Because vav-lacZ transgene expression decreased
progressively,11 we monitored hCD4 in peripheral blood as
individual vav-hCD4 animals aged. Mice aged 1 year yielded
profiles (Fig 7A and B) indistinguishable
from those they had given at 3 weeks of age. Indeed, in striking
contrast to vav-lacZ expression (Fig 7C), none of the 8 vav-hCD4 lines showed a decrease in either the proportion of
hCD4+ blood cells or the average level of expression (mean
fluorescence intensity per cell).
View larger version (29K):
[in this window]
[in a new window]
| Fig 7.
Stability of vav-hCD4 transgene expression. hCD4
levels on viable peripheral blood leukocytes from individual HS21/45
mice, of 2 independent lines, at more than 1 year of age (A and B). The
proportion of cells with transgene-associated fluorescence above that
from a nontransgenic littermate (dotted and shaded histogram) is given.
(C) Maintenance of full transgene activity in individual vav-hCD4
mice (open symbols, representing 3 different lines) versus the
decrease in mice of a typical vav-lacZ line (solid symbols).
|
|
| |
DISCUSSION |
A potent pan-hematopoietic promoter.
Our results indicate that the cluster of 5 HS sites in the 5'
portion of the vav gene (Fig 1) constitute its major regulatory elements. A transgene bearing 4 of these sites, as well as 1 including the distal upstream HS3 site, efficiently drove hCD4 expression in
approximately 80% of primary mice. Expression was stable for as long
as analyzed (6 to 18 months; Fig 7), with no evidence of variegation or
decrease in any of 8 independent progeny lines. In adult animals, the
transgenes were active in every nucleated hematopoietic cell type
examined, including B and T lymphocytes, neutrophils, monocytes,
megakaryocytes, eosinophils, and erythroid cells. The highest level
appeared in lymphocytes, eosinophils, and megakaryocytes, whereas
monocytes and neutrophils had an intermediate level and erythroid cells
had the lowest level (Table 1). In accord with these results, vav
expression is reported to be particularly high in lymphocytes
and megakaryocytes,4 but insufficient data are available to
say whether the expression of the vav-hCD4 transgene entirely
mirrors that of vav.
Significantly, we found that all blast cells and all clonogenic
progenitors assayed bore hCD4, including those committed to the
neutrophil, monocyte/macrophage, eosinophil, and megakaryocyte lineages
(Table 2). Although erythroid precursors were not tested, they are
likely to be positive, because the great majority of cells bearing
TER119, a marker found on erythroid precursors, also displayed hCD4
(Fig 3), and in general the immature cells of the various lineages
seemed to have the highest levels of transgene expression. Indeed, high
levels appeared on all the more ancestral preprogenitors, which yield
blast cell progeny (Table 2). Thus, the vav promoter was very
active in cells near the apex of the hematopoietic hierarchy as well as
in all their mature progeny.
We found no evidence for expression in nonhematopoietic cell types (Fig
6). The absence of expression in testis (Fig 6D) might seem surprising,
because an isoform of vav is found there, but it arises from a
separate promoter 1 kb upstream of exon 2,6 absent from our
transgenes (Fig 1A).
In the fetus, hCD4 appeared on definitive erythroid cells and, at
higher levels, on minor nonerythroid populations such as that bearing
Mac-1 (Fig 5). Because the multipotential progenitors in fetal liver
are Mac1+,27 the presence of hCD4 on the vast
majority of fetal Mac1+ cells (Fig 5B) may indicate that
the vav promoter is also active in fetal multipotential
progenitors. Our failure to detect transgene expression in primitive
erythrocytes (Fig 5D) is consistent with vav itself being
undetectable in the embryo before E11.5.4,5 Nevertheless,
the blood did contain a small population of hCD4+ cells
even at E10.5 (Fig 5F). Perhaps the vav promoter functions in
stem or progenitor cells, even during primitive hematopoiesis, but is
turned off during the differentiation of primitive erythrocytes.
Thus, with the exception of primitive erythrocytes, the vav
vectors appear to function in a pan-hematopoietic fashion. This conclusion has recently been confirmed with vav-bcl-2 mice,
which exhibit effects in multiple hematopoietic lineages (our
unpublished results). The levels of Bcl-2 in their
lymphocytes indicate that the vav promoter is at least as
potent as the well-tested Ig enhancer (Eµ) transgenic
vectors.28
Because all 5 vav HS sites appear in several hematopoietic
lineages and not in fibroblasts,11 these sites may normally
act together, both to open the surrounding chromatin within
hematopoietic stem cells and to maintain this open state during
subsequent differentiation. That would explain why the vav
elements were potent when integrated into chromatin but impotent in
conventional promoter/enhancer assays not involving
integration.11 The vav regions used here evidently
can function in most chromosomal positions, and the holo-complex may be
akin to a locus control region,12,13 although how an LCR
functions remains uncertain.15,29
Inactivation by the prokaryotic reporter gene.
In striking contrast to the present results, all 18 independent
vav-lacZ lines exhibited variegation,11 and the
percentage of -gal+ cells decreased as animals aged (Fig
7C) and as lymphocytes matured, whereas no expression was detectable in
nonlymphoid hematopoietic cells. Although a large array of transgenes
can provoke variegation,30 the vav-lacZ lines had
very low copy numbers,11 as did the vav-hCD4 lines
(not shown), and the uniformity of the lacZ results makes insertion into heterochromatin31 an unlikely general
explanation. Thus, the reporter itself must be largely responsible.
Comparison with the present results suggests that lacZ both
prevented expression in several hematopoietic lineages and in
lymphocytes induced sporadic irreversible silencing of the transgene.
Although variegation can occur with transgenes entirely of vertebrate
origin,31 lacZ has featured in many studies
describing variegation,14,32-36 and it may well
have exacerbated or even induced the heterocellularity. Most likely,
only certain promoters are affected, because there are mice in which
lacZ appears to be expressed in all cells.37
A review on lacZ as a transgenic reporter38
concluded that "its postnatal in vivo expression has been
unreliable and disappointing." Its erratic performance in long-term
experiments would be explicable if the bacterial sequences
stochastically inactivated certain linked promoters, so that the
proportion of nonexpressing cells increased with time or number of cell
divisions. The inactivation might be due to stable repressed complexes,
heterochromatinization, and/or methylation. It seems relevant that the
3.2-kb lacZ sequence contains numerous potential binding sites
for mammalian transcription factors, including ubiquitous ones such as
SP-1, and hematopoietic factors such as GATA-1, Myb, and Scl. Complexes
of such factors on the lacZ DNA sequence could
interfere with proper assembly on linked mammalian regulatory elements.
LacZ also contains numerous CG sequences, which are
potential mammalian methylation sites, although
methylation is usually thought to consolidate rather than to create a
silent state. The deleterious effects of lacZ probably are
typical of many prokaryotic sequences, because plasmid sequences have
long been known to compromise transgene activity.38 For
long-term in vivo studies, a more reliable cell-by-cell reporter may be
provided by a mammalian cell surface marker such as hCD4, which
functioned appropriately here, or the humanized Green Fluorescent Protein.39
Potential of multilineage hematopoietic targeting.
If, as seems likely, the vav promoter is active in the
hematopoietic stem cell, vav-hCD4 mice might aid the
recognition and isolation of those very rare cells by, for example,
marking the first intraembryonic stem cells.25 Because the
other available transgenic promoters for hematopoietic cells function
in only 1 or 2 lineages, or at particular differentiation stages, the ability of the vav promoter to target expression throughout the compartment should facilitate studies on many aspects of hematopoiesis. For instance, vav-driven expression of transcription factors
implicated in lineage commitment1 should clarify whether
they actually impose lineage specification. Similarly, because certain
leukemias are thought to arise in stem cells, a vav transgenic
vector should facilitate tests on putative oncogenes. Conversely, a
vav-driven Cre recombinase might allow hematopoietic-specific
knockout of genes with wider essential function. Finally, vav
regulatory sequences may prove useful tools in the search for genes
that induce commitment of mesodermal cells to hematopoietic development.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs Suzanne Cory and Andrew Elefanty for
comments on the manuscript, Drs Elefanty and Lorraine Robb for advice
on fetal analysis, Li-Chen Zhang and Sandra Mifsud for technical
assistance, Adrian Mifsud and Jodie de Winter for animal husbandry, Dr
Andreas Strasser for antibodies, Dr Bernd Hentsch for a cloned
vav sequence, and Jeanette Birtles for preparation of the manuscript.
| |
FOOTNOTES |
Submitted January 25, 1999; accepted May 20, 1999.
Supported by the National Health and Medical Research Council, Canberra
(Reg. Key 973002) and the US National Cancer Institute (CA43540 and CA80188).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jerry M. Adams, PhD, The Walter and Eliza
Hall Institute of Medical Research Post Office, Royal Melbourne
Hospital, Victoria, 3050 Australia; e-mail: adams{at}wehi.edu.au.
| |
REFERENCES |
1.
Shivdasani RA, Orkin SH:
The transcriptional control of hematopoiesis.
Blood
87:4025, 1996[Free Full Text]
2.
Katzav S, Martin Zanca D, Barbacid M:
vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells.
EMBO J
8:2283, 1989[Medline]
[Order article via Infotrieve]
3.
Adams JM, Houston H, Allen J, Lints T, Harvey R:
The hematopoietically expressed vav proto-oncogene shares homology with the dbl GDP-GTP exchange factor, the bcr gene and a yeast gene (CDC24) involved in cytoskeletal organisation.
Oncogene
7:611, 1992[Medline]
[Order article via Infotrieve]
4.
Bustelo XR, Rubin SD, Suen K-L, Carrasco D, Barbacid M:
Developmental expression of the vav protooncogene.
Cell Growth Differ
4:297, 1993[Abstract]
5.
Keller G, Kennedy M, Papayannopoulou T, Wiles MV:
Hematopoietic commitment during embryonic stem cell differentiation in culture.
Mol Cell Biol
13:473, 1993[Abstract/Free Full Text]
6.
Okumura K, Kaneko Y, Nonoguchi K, Nishiyama H, Yokoi H, Higuchi T, Itoh K, Yoshida O, Miki T, Fujita J:
Expression of a novel isoform of vav, vav-t, containing a single SRC homology 3 domain in murine testicular germ cells.
Oncogene
14:713, 1997[Medline]
[Order article via Infotrieve]
7.
Zmuidzinas A, Fischer K-D, Lira SA, Forrester L, Bryant S, Bernstein A, Barbacid M:
The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro.
EMBO J
14:1, 1995[Medline]
[Order article via Infotrieve]
8.
Crespo P, Schuebel KE, Ostrom AA, Gutkind JS, Bustelo XR:
Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the Vav proto-oncogene product.
Nature
385:169, 1997[Medline]
[Order article via Infotrieve]
9.
Turner M, Mee PJ, Walters AE, Quinn ME, Mellor AL, Zamoyska R, Tybulewicz VLJ:
A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes.
Immunity
7:451, 1997[Medline]
[Order article via Infotrieve]
10.
Zhang R, Alt FW, Davidson L, Orkin SH, Swat W:
Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene.
Nature
374:470, 1995[Medline]
[Order article via Infotrieve]
11.
Ogilvy S, Elefanty AG, Visvader J, Bath ML, Harris AW, Adams JM:
Transcriptional regulation of vav, a gene expressed throughout the hematopoietic compartment.
Blood
91:419, 1998[Abstract/Free Full Text]
12.
Martin DIK, Whitelaw E:
The vagaries of variegating transgenes.
Bioessays
18:919, 1996[Medline]
[Order article via Infotrieve]
13.
Kioussis D, Festenstein R:
Locus control regions: Overcoming heterochromatin-induced gene inactivation in mammals.
Curr Opin Genet Dev
7:614, 1997[Medline]
[Order article via Infotrieve]
14.
Walters MC, Magis W, Fiering S, Eidemiller J, Scalzo D, Groudine M, Martin DIK:
Transcriptional enhancers act in cis to suppress position-effect variegation.
Genes Dev
10:185, 1996[Abstract/Free Full Text]
15.
Higgs DR:
Do LCRs open chromatin domains?
Cell
95:299, 1998[Medline]
[Order article via Infotrieve]
16.
Moebius U, Pallai P, Harrison SC, Reinherz EL:
Delineation of an extended surface contact area on human CD4 involved in class II major histocompatibility complex binding.
Proc Natl Acad Sci USA
90:8259, 1993[Abstract/Free Full Text]
17.
Maddon PJ, Littman DR, Godfrey M, Maddon DE, Chess L, Axel R:
The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: A new member of the immunoglobulin gene family.
Cell
42:93, 1985[Medline]
[Order article via Infotrieve]
18.
Glaichenhaus N, Shastri N, Littman DR, Turner JM:
Requirement for association of p56lck with CD4 in antigen-specific signal transduction in T cells.
Cell
64:511, 1991[Medline]
[Order article via Infotrieve]
19.
Takebe Y, Seiki M, Fujisawa J-I, Hoy P, Yokota K, Arai K-I, Yoshida M, Arai N:
SR promoter: An efficient and versatile mammalian cDNA expression system composed of the Simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat.
Mol Cell Biol
8:466, 1988[Abstract/Free Full Text]
20.
Hogan B, Beddington R, Costantini F, Lacy E:
Manipulating the Mouse Embryo. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1994.
21.
Unkeless JC:
Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.
J Exp Med
150:580, 1979[Abstract/Free Full Text]
22.
Strasser A, Harris AW, Cory S:
Bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship.
Cell
67:889, 1991[Medline]
[Order article via Infotrieve]
23.
Nolan GP, Fiering S, Nicholas F, Herzenberg LA:
Fluorescence-activated cell analysis and sorting of viable mammalian cells based on -D-galactosidase activity after transduction of Escherichia coli lacZ.
Proc Natl Acad Sci USA
85:2603, 1988[Abstract/Free Full Text]
24.
Metcalf D:
Clonal Cultures of Haemopoietic Cells: Techniques and Applications. Amsterdam, The Netherlands, Elsevier, 1984.
25.
Dzierzak E, Medvinsky A:
Mouse embryonic hematopoiesis.
Trends Genet
11:359, 1995[Medline]
[Order article via Infotrieve]
26.
Trimborn T, Gribnau J, Grosveld F, Fraser P:
Mechanisms of developmental control of transcription in the murine - and -globin loci.
Genes Dev
13:112, 1999[Abstract/Free Full Text]
27.
Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL:
The purification and characterization of fetal liver hematopoietic stem cells.
Proc Natl Acad Sci USA
92:10302, 1995[Abstract/Free Full Text]
28.
Adams JM, Cory S:
Transgenic models of tumor development.
Science
254:1161, 1991[Abstract/Free Full Text]
29.
Grosveld F:
Activation by locus control regions?
Curr Opin Genet Dev
9:152, 1999[Medline]
[Order article via Infotrieve]
30.
Garrick D, Fiering S, Martin DI, Whitelaw E:
Repeat-induced gene silencing in mammals.
Nat Genet
18:56, 1998[Medline]
[Order article via Infotrieve]
31.
Festenstein R, Tolaini M, Corbella P, Mamalaki C, Parrington J, Fox M, Miliou A, Jones M, Kioussis D:
Locus control region function and heterochromatin-induced position effect variegation.
Science
271:1123, 1996[Abstract]
32.
Guy L-G, Kothary R, De Repentigny Y, Delvoye N, Ellis J, Wall L:
The -globin locus control region enhances transcription of but does not confer position-independent expression onto the lacZ gene in transgenic mice.
EMBO J
15:3713, 1996[Medline]
[Order article via Infotrieve]
33.
Robertson G, Garrick D, Wu W, Kearns M, Martin D, Whitelaw E:
Position-dependent variegation of globin transgene expression in mice.
Proc Natl Acad Sci USA
92:5371, 1995[Abstract/Free Full Text]
34.
Sutherland HGE, Martin DIK, Whitelaw E:
A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.
Mol Cell Biol
17:1607, 1997[Abstract]
35.
McDevitt MA, Fujiwara Y, Shivdasani RA, Orkin SH:
An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice.
Proc Natl Acad Sci USA
94:7976, 1997[Abstract/Free Full Text]
36.
Akagi K, Sandig V, Vooijs M, Van der Valk M, Giovannini M, Strauss M, Berns A:
Cre-mediated somatic site-specific recombination in mice.
Nucleic Acids Res
25:1766, 1997[Abstract/Free Full Text]
37.
Soriano P:
Generalized lacZ expression with the ROSA26 Cre reporter strain.
Nat Genet
21:70, 1999[Medline]
[Order article via Infotrieve]
38.
Cui C, Wani MA, Wight D, Kopchick J, Stambrook PJ:
Reporter genes in transgenic mice.
Transgenic Res
3:182, 1994[Medline]
[Order article via Infotrieve]
39.
Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y:
`Green mice' as a source of ubiquitous green cells.
FEBS Lett
407:313, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
A. Banerjee, R. Grumont, R. Gugasyan, C. White, A. Strasser, and S. Gerondakis
NF-{kappa}B1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms
Blood,
December 15, 2008;
112(13):
5063 - 5073.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Khurana and A. Mukhopadhyay
Hematopoietic Progenitors from Early Murine Fetal Liver Possess Hepatic Differentiation Potential
Am. J. Pathol.,
December 1, 2008;
173(6):
1818 - 1827.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. He, H. Hu, R. Braren, S.-Y. Fong, A. Trumpp, T. R. Carlson, and R. A. Wang
c-myc in the hematopoietic lineage is crucial for its angiogenic function in the mouse embryo
Development,
July 15, 2008;
135(14):
2467 - 2477.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Salmond, L. McNeill, N. Holmes, and D. R. Alexander
CD4+ T cell hyper-responsiveness in CD45 transgenic mice is independent of isoform
Int. Immunol.,
July 1, 2008;
20(7):
819 - 827.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Slape, Y. W. Lin, H. Hartung, Z. Zhang, L. Wolff, and P. D. Aplan
NUP98-HOX Translocations Lead to Myelodysplastic Syndrome in Mice and Men
J Natl Cancer Inst Monographs,
July 1, 2008;
2008(39):
64 - 68.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Tiedt, H. Hao-Shen, M. A. Sobas, R. Looser, S. Dirnhofer, J. Schwaller, and R. C. Skoda
Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice
Blood,
April 15, 2008;
111(8):
3931 - 3940.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ogilvy, R. Ferreira, S. G. Piltz, J. M. Bowen, B. Gottgens, and A. R. Green
The SCL +40 Enhancer Targets the Midbrain Together with Primitive and Definitive Hematopoiesis and Is Regulated by SCL and GATA Proteins
Mol. Cell. Biol.,
October 15, 2007;
27(20):
7206 - 7219.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Caudell, Z. Zhang, Y. J. Chung, and P. D. Aplan
Expression of a CALM-AF10 Fusion Gene Leads to Hoxa Cluster Overexpression and Acute Leukemia in Transgenic Mice
Cancer Res.,
September 1, 2007;
67(17):
8022 - 8031.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. P. Smith, M. L. Bath, D. Metcalf, A. W. Harris, and S. Cory
MYC levels govern hematopoietic tumor type and latency in transgenic mice
Blood,
July 15, 2006;
108(2):
653 - 661.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Wiesner, J. M. Jones, D. E. Hasz, and D. A. Largaespada
Repressible transgenic model of NRAS oncogene-driven mast cell disease in the mouse
Blood,
August 1, 2005;
106(3):
1054 - 1062.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-W. Lin, C. Slape, Z. Zhang, and P. D. Aplan
NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia
Blood,
July 1, 2005;
106(1):
287 - 295.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Stadtfeld and T. Graf
Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing
Development,
January 1, 2005;
132(1):
203 - 213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Coultas, P. Bouillet, E. G. Stanley, T. C. Brodnicki, J. M. Adams, and A. Strasser
Proapoptotic BH3-Only Bcl-2 Family Member Bik/Blk/Nbk Is Expressed in Hemopoietic and Endothelial Cells but Is Redundant for Their Programmed Death
Mol. Cell. Biol.,
February 15, 2004;
24(4):
1570 - 1581.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ogilvy, C. Louis-Dit-Sully, J. Cooper, R. L. Cassady, D. R. Alexander, and N. Holmes
Either of the CD45RB and CD45RO Isoforms Are Effective in Restoring T Cell, But Not B Cell, Development and Function in CD45-Null Mice
J. Immunol.,
August 15, 2003;
171(4):
1792 - 1800.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. S. Radomska, D. A. Gonzalez, Y. Okuno, H. Iwasaki, A. Nagy, K. Akashi, D. G. Tenen, and C. S. Huettner
Transgenic targeting with regulatory elements of the human CD34 gene
Blood,
December 15, 2002;
100(13):
4410 - 4419.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ogilvy, D. Metcalf, C. G. Print, M. L. Bath, A. W. Harris, and J. M. Adams
Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival
PNAS,
December 21, 1999;
96(26):
14943 - 14948.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION