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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1274-1282
HEMATOPOIESIS
From the Department of Medicine, Division of Hematology, University
of Washington, Seattle, WA and the Department of Biochemistry and
Molecular Biology, University of Kansas, Kansas City,
KS.
A substantial body of published data suggests activation of
lineage-specific genes in multipotential hemopoietic cells before their
unilineage commitment. Because the behavior and plasticity of cells
isolated in vitro away from microenvironmental constraints exercised in
vivo may be altered, one wonders whether similar findings can be
observed in a physiologic setting in vivo. We used a transgenic mouse
model harboring human micro LCR together with
Functional hemopoietic cells of different lineages are
constantly replenished by a pool of lineage-committed progenitor cells, which in turn are generated on demand by multipotent stem cells. The
ability of the latter cells to become differentiated, together with
their ability to self-renew, constitutes the unique fundamental function of stem cells.1 Understanding the molecular
processes that underlie both the self-renewal process and the
differentiation decisions in the hemopoietic system is not only of
biologic significance, it has implications in transplantation, gene
therapy, and disease pathogenesis (ie, leukemias). Therefore,
parameters that influence the decision-making process by stem cells are
under intense investigation. Based on numerous cellular or molecular
studies conducted thus far, it has been suggested that commitment of
adult hemopoietic stem cells to a specific lineage is an intrinsic,
stochastic process,2-10 though the possibility of external
influence at some stem cell-progeny level is still actively
debated.11 Early data generated by Ogawa et al4
on the fates of 2 daughter cells separated in vitro have suggested that
lineage selection is accomplished not by acquisition of new markers but
by sequential restriction of potentialities of multipotent cells in a
stochastic fashion. Other variations of this theory suggest that the
restriction of multipotentiality is accomplished not stochastically but
in a predetermined, hierarchical fashion.12 These views
have gained new life by recent molecular analyses of early
multipotential cells.5-9 The goal of these studies was to
attempt to analyze the molecular expression profiles of multipotential
or uncommitted cells to provide a glimpse at the molecular basis of
lineage commitment. Initial studies were conducted in cell lines
thought to represent multipotential cells, especially the ones capable
of responding to physiologic stimuli with downstream differentiation to
more than 1 lineage.13-15 Various genes with distinctive
lineage affiliations were found to be present in these cells at the
chromatin level and at the transcriptional level of these genes.
Furthermore, it was found that the expression and/or
transcription of both of these genes is either accentuated or is
down-regulated, depending on the lineage pathway chosen. Thus,
commitment to nonerythroid (ie, monocytic or lymphocytic) pathway shuts
down expression of the epo-receptor gene or of the erythroid
transcription factor GATA-1, whereas a dramatic up-regulation of
these genes is seen with erythroid differentiation.
However, many of the cell lines studied continuously proliferate in
vitro, unlike true stem cells, and show spontaneous downstream differentiation to limited lineage pathways.16 Therefore,
many of the data addressing characteristics of primitive hemopoietic cells at the population level may simply represent differences among
subpopulations of more differentiated cells in these cell lines. These
concerns were greatly alleviated when single cells from cell lines were
tested by sensitive reverse transcription-polymerase chain reaction
(RT-PCR) approaches.9 These important studies showed that
low levels of transcription of lineage-affiliated genes, such as globin
or myeloperoxidase, are coexpressed within individual cells. The survey
of genes investigated included lineage-affiliated transcription
factors, such as GATA-1, growth-factor receptors (GM-CSF, kit, M-CSF,
or Epo receptors), or lineage-specific genes (globin). In general, the
expression levels were low, and no lineage-affiliated proteins were
detected in these studies. In contrast to expectations, transcriptional
factors characteristic of a single lineage and lineage-specific genes
(ie, globin, GATA-1, or epo-R) were not expressed in tandem in these
studies. Most important, coactivation of genes of multiple lineages
within single cells was also found when primary CD34+ cells were
examined instead of FDCP cells.
Several arguments can be raised with these previously published data.
They concern cells adapted to in vitro conditions, cells that are
continuously proliferating and that have potential activation of
certain molecular pathways. These cells may have options that may not
be exercised in vivo. In addition, primary cells, because of methods of
isolation or of conditions under which they are kept in vitro to ensure
viability, may not be subjected to negative constraints exercised in
vivo by the hemopoietic microenvironment and as a result may display a
certain state of activation in vitro. Because the hemopoietic
microenvironment cannot be faithfully imitated in vitro, it is unclear
whether the results can be directly extrapolated in vivo. Furthermore,
in other studies17,18 lineage-affiliated genes were not
found to be activated in early cells, though the sensitivity of methods
used may have been the problem in these studies.
Because the priming of lineage-specific genes in precommitted cells has
been thus far only an in vitro phenomenon, in the current study we
explored whether it could also be observed in vivo. To do so, we used a
transgenic murine model in which a µLCR-driven Synthesis of µLCR-
Generation of µLCR- FACS-Gal assay
X-gal assay Mononuclear cells from bone marrow, fetal liver, or blood were rinsed in phosphate-buffered saline (PBS), fixed for 5 minutes on ice in a fixative solution of 1% formaldehyde and 0.5% glutaraldehyde in PBS, rinsed in detergent buffer (PBS, 0.02% NP40, 0.01% deoxycholate, 2 mmol/L MgCl2), and stained at 37°C in the dark for 3 to 4 hours. Staining solution contained 1 mg/mL X-gal [5-bromo-4-chloro-3-indolyl- -D-galacto-pyranoside], 5 mmol/L
K3Fe(CN)6, 5 mmol/L
K4Fe(CN)6, and 1 mmol/L EGTA (ethylene glycol-bis(-aminoethyl ether) N, N, N', N'-tetraacetic acid) in
detergent buffer.24 Frozen sections were fixed and stained similarly. Whole organs were fixed for 30 minutes, processed as above,
and stained overnight. Plasma clots were flattened on a slide with
filter paper, fixed for 5 minutes, and stained as above for exactly 1 hour, rinsed, fixed in 3% glutaraldehyde, rinsed in distilled
H20, then stained with benzidine and hematoxylin as
previously described.25
Colony-forming unit-C assays Colony-forming unit (CFU)-C assays were performed using a mixture containing 30% fetal bovine serum (FBS; Intergen, Purchase, NY), 1% bovine serum albumin (BSA; Intergen, Purchase, NY), 0.1 mmol/L 2-mercaptoethanol (Sigma, St. Louis, MO), 5 U/mL recombinant human erythropoietin (EPO; Genetics Institute, Cambridge, MA), 10% vol/vol mouse IL-3 culture supplement (Collaborative Biomedical Products, Bedford, MA), 5% pokeweed mitogen spleen cell-conditioned medium, 100 ng/mL recombinant murine stem cell factor (Peprotech, Rocky Hill, NJ), and 1.2% methyl cellulose (Fisher Scientific, Fairlawn, NJ). Plasma clot cultures contained the above mixture (without methyl cellulose), 2 mmol/L CaCl2, 0.25 U/mL bovine thrombin (Sigma), and 10% bovine citrated plasma (Animal Technologies, Tyler, TX). Medium components were made up in Iscove's modified Dulbecco's medium (Mediatech, Hernden, VA). Cells were plated as previously described26 and counted after 7 to 8 days.Colony-forming unit-S assays Fetal liver or bone marrow mononuclear cells from the LacZ mice were subjected to red cell lysis, labeled with TER119 antibody followed by antirat IgG-phycoerythrin, then stained with FDG and sorted using a Becton Dickinson FACStar Plus. 5 × 104 sorted FDG(+) or FDG( ) cells were then injected into the tail veins of sublethally irradiated SCID/NOD recipient mice. (Because of
histocompatability problems, no syngeneic mice could be used.) Mice
were killed 8 or 12 days later, their spleens were excised, and CFU-S
were counted in situ or individually dissected out of the spleens,
fixed, and stained with X-gal. Some spleens were mounted in OCT
embedding medium (Tissue-Tek, Torrance, CA), and frozen sections were
then fixed and stained with X-gal to assess positivity of CFU-S.
LacZ activation in the developing fetus Histochemical X-gal staining was carried out using either whole embryos or yolk sacs or using cell suspensions prepared from yolk sac, fetal blood, or fetal liver. Whole embryo staining revealed LacZ expression in blood vessels and hemopoietic organs (yolk sac, fetal liver, and bones; Figure 2A). This pattern of expression, restricted to hemopoietic cells and organs, was reminiscent of results published previously when similar constructs were used.27,28 Circulating embryonic or definitive erythroblasts and red cells present in blood at days 11 to 12 days post coitus were heavily labeled. However, white cells present in the same samples were not labeled (Figure 2B). Cell preparations from fetal liver also showed heavy labeling obscuring even the presence of white, unlabeled cells. High proportions of erythroblasts in these preparations were responsible for the intense labeling. To secure the identity of many small cells labeled with LacZ, we used a double labeling approach in which benzidine staining was combined with X-gal staining. All erythroid cells were benzidine positive, and the combination of gold (from benzidine staining) and green (for X-gal staining) gave these cells a deep bronze appearance (Figures 2C, 2D).
LacZ activation in differentiated erythroid and megakaryocytic cells To test the presence of X-gal activity among differentiated cells of several lineages, peripheral blood cells or bone marrow mononuclear cells from adult mice were labeled in suspension by the X-gal technique, as described in the "Materials and methods" section. After staining, cytocentrifuge preparations were made to score individual cells. Among peripheral blood cells, the only cells with X-gal activity were the red cells. The proportion of positive red cells among 26 mice tested was between 84% and 100%, and among positive cells there were visible differences in the intensity of staining. Timed incubation for X-gal (between 15 minutes and 2 hours) exaggerated these differences. In 1 sample the proportion of X-gal-positive cells was approximately 60% at 15 minutes, but virtually all cells were positive at 2 hours. In bone marrow preparations, erythroblasts at all maturation stages were positive, and the majority appeared strongly positive. In addition to erythroid cells, megakaryocytes on the smears were positive, again with great differences in intensity of staining.LacZ activation in BFUe and CFU-meg-derived progeny To ensure that only differentiated erythroid or megakaryocytic cells at all stages of maturation were positive, we cultivated cell suspensions from yolk sac, fetal liver, or adult bone marrow in semi-solid (methyl cellulose or plasma clot) media and tested the X-gal positivity in the in vitro progeny of all clonogenic progenitors. Methyl cellulose culture plates were evaluated in situ for all types of colonies present on the basis of morphologic criteria and then were stained with the X-gal for evaluation of total "blue" colonies versus "non-blue" colonies. Plasma clots were evaluated after they were fixed and stained as described in "Materials and Methods." The proportion of erythroid bursts, especially those of mixed erythroid and megakaryocytic colonies, was higher in yolk sac or fetal liver than in adult bone marrow (Ery/Meg colonies: yolk sac, 8.7 ± 1.8/105 cells plated; fetal liver, 3.9 ± 1.8/105; adult bone marrow, less than 0.01). The total number of blue colonies was sometimes slightly lower than the total number of erythroid/megakaryocytic colonies (data not shown). This difference was as high as 5% in fetal samples and 30% in adult samples. In other words, more than 92% of erythroid and megakaryocytic colonies were strongly positive for X-gal in fetal samples, whereas in adult samples an average of 67% of the colonies were strongly X-gal positive. Whether colonies scored as negative were totally negative or whether some weakly positive cells were present was not assessed because these methylcellulose counts were performed only under the dissecting microscope. In flattened plasma clot cultures, in addition to apparently negative bursts, bursts displaying mixed positive and negative cells were clearly present (Figure 2D), especially in adult bone marrow cells. Heterogeneity in staining was also seen in megakaryocytic colonies in the same cultures.FDG(+) cells include progenitors of all lineages In the previous sections we described that differentiated erythroid and megakaryocytic cells in vivo, and the ones generated in vitro by committed progenitors in culture were positive in X-gal staining. To test whether these positive cells were generated after terminal commitment to these 2 lineages or whether the activation of LacZ was already present in precommitted cells, we depleted bone marrow or fetal liver cells of erythroid cells (by removing TER119+ cells) and labeled the resultant TER119() cells with the
fluorescent LacZ substrate FDG, as described in "Materials and Methods." FDG labeling is a highly sensitive way to assess even
low levels of LacZ activation. FDG-stained bone marrow cells from nontransgenic mice were used as negative control samples. Positive
controls were FDG-stained bone marrow cells from Rosa--geo mice in
which all cells are X-gal positive except red cells. In fetal livers
TER-119(+) cells displayed similar positivity to Rosa--geo cells,
and these were also used as positive controls. The proportion of FDG(+)
cells among TER119() fetal liver samples was very high (see
Table 1) and showed a broad spectrum of positivity (only
2% to 13% of cells were FDG(); Table 1, Figure
3). By contrast, the
proportion of FDG(+) cells among adult bone marrow/TER119() cells was much lower; in 4 bone marrow samples, the proportion of
FDG() cells was 36% to 75% (Figure
4). In bone marrow samples of 2 newborns,
proportions of FDG(+) and TER119() cells were 50.2% and 34.2%.
Not only was the proportion of FDG(+)/TER119() cells lower in
bone marrow samples when compared with fetal liver cells, the mean
fluorescence intensity (Figure 3 vs Figure 4) was also much lower.
After the distribution of FDG positivity was determined, populations
highly positive for FDG, displaying similar positivity with
Rosa--geo controls (Figure 3A), or populations with intermediate FDG
positivity and populations totally negative for FDG were sorted and
subjected to clonogenic cultures to determine the types of colonies
present in these fractions. Results from these analyses are listed in
Tables 1 and 2. Three fetal liver samples
and 4 bone marrow samples were analyzed. From fetal liver samples,
clearly the majority of colonies generated were among the FDG(+)
fractions (considering the frequency among plated cells and the
frequency of FDG(+) among total cells). Most interesting, not
only the erythroid/megakaryocytic progenitors but also the majority of
myeloid progenitors, CFU-GM, were found in these positive fractions,
though progeny of the latter was negative for X-gal in vitro.
Independent evaluation of X-gal positive (blue) versus X-gal negative
(non-blue) colonies secured these findings (ie, number of blue colonies
was similar to the number of erythroid and megakaryocytic colonies). In
addition to informative findings from FDG(+) cells, the types of
colonies generated from FDG() cells were also of interest.
Erythroid bursts with X-gal(+) progeny were also generated from these
fractions. Although LacZ positivity was not detected in
progenitors, the gene was not irreversibly silenced; its progeny
displayed full activation of LacZ during erythroid
differentiation. Highly positive FDG fractions in 1 liver sample
(Figure 3A) generated virtually only CFUe. The degree of positivity in
this fraction was equivalent to that of TER119(+) cells or the
Rosa--geo cells (Figure 3A). Therefore, it may not be
surprising that this fraction showed only late erythroid progenitors and precursors.
CFU-S12 are generated from transplantation of FDG(+) cells One bone marrow and one fetal liver sample were used for CFU-S12 studies (Table 3). The samples were simultaneously labeled for TER119-PE and for FDG-FITC. After the TER119(+) cells were excluded, the population was divided into FDG(+) and FDG() cells. After 5 × 104 cells
[FDG(+) or FDG()] were injected into each mouse, CFU-S12 colonies were counted in whole spleens before or after X-gal staining. Forty colonies were counted in 5 mice given FDG() cells from the
bone marrow sample. Four mice given FDG(+) cells generated 39 colonies.
(In the 5th mouse, colonies were too confluent for accurate counting.)
Because the SCID/NOD mice were only sublethally irradiated, CFU-S12 in
these mice generated from FDG(+) or FDG() cells could represent
just endogenous CFU-S. Spleens were then subjected to X-gal staining.
The diffuse green color in these spleens (because of the presence of
red cells and erythroblasts matured from CFU-S8) obscured the true
positivity of CFU-S. Therefore, a different approach was used in the
next experiment. From 1 fetal liver sample, only the FDG(+) cells were
injected into 8 mice. Four mice were killed at day 8, and CFU-S8 were
counted (there were 36 CFU-S, and most appeared blue after staining of
frozen spleen sections with X-gal; Figure 2E). From 4 mice killed at day 12, 26 colonies were counted, and 17 of these were individually dissected to be X-gal stained separately in vitro. 70% of colonies individually stained showed more than 20% positive cells in each. (Colonies with less than 20% positive cells were considered negative.) Spleen sections stained for X-gal (Figure 2E) were also prepared. These
sections showed either fairly uniform X-gal positivity (Figure 2E) or
scattered positive cells among each CFU-S colony. Thus, in these
experiments, we showed that CFU-S, like CFC, were present among FDG(+)
cells, though the fraction of total CFU-S12 could not be calculated
with accuracy (because of the nature of recipient mice).
Hemopoietic cell subsets with a stem cell phenotype are FDG positive Histocompatibility problems precluded long-term repopulation experiments using FDG(+) or FDG() cells. Using a surrogate
approach, we evaluated the LacZ activation in subsets of stem
cells previously shown to include long-term repopulating cells, such as
Lin/kit+/CD34+ or Lin/Sca1+/kit+. One fetal liver
sample and 1 bone marrow sample from a newborn were used for these
studies. The proportion of Lin/kit+/34+ subset in fetal liver
was 11.4%, whereas the proportion of Lin/Sca1+/kit+
cells was 15.7%. The proportions in newborn bone marrow were 3.5% and
7.1%, respectively. However, the great majority of either
Lin/Sca1+/kit+ and Lin/kit+/CD34+ in both samples
were FDG(+) [Figure 5], suggesting that
LacZ activation may indeed start early at the multipotential
cell level, possibly in a proportion of long-term repopulating cells.
Our experiments thus far do not allow any conclusions regarding the
level of activation in long-term repopulating cells. The latter can
only be reliably tested with transplantations of FDG(+) stem
cell subsets in histocompatible recipients. Nevertheless, we
have evaluated SCID/NOD mice that received FDG(+) cells 5 weeks after
transplantation. Two such mice showed approximately 50% of their red
cells X-gal (+) (Figure 2F). In addition, bone marrow from 1 mouse was
cultured in plasma clots to assess the presence of X-gal (+) colonies.
This marrow yielded a total of 229 ± 19 CFU-C/105
plated cells. The total erythroid bursts were 51 ± 4, CFU-Meg were
20 ± 3, CFU-MixEry + Meg were 7 ± 2, and CFU-GM were 151 ± 18/105 cells plated. Fifty-five colonies
were blue ie, approximately 73% of the total erythroid and
megakaryocytic colonies were X-gal (+).
Although previous data showing simultaneous expression of
lineage-affiliated genes in multipotential cells from established cell
lines or in primary human CD34+ cells are compelling,9 the
concept of activation of lineage-restricted genes in cells before their
overt unilineage commitment has not been tested in an in vivo setting.
We elected to explore this concept in vivo using transgenic mice,
carrying the LCR-driven Erythroid and megakaryocytic cells display high levels of LacZ activity As expected from previous studies using similar constructs,24, 27,28 we found significant expression of LacZ on erythroid cells. However, in contrast to previously reported data, we found X-gal-positive cells among cells of megakaryocytic lineage. Megakaryocytes generated in vivo or induced from CFU-Meg during clonogenic cultures in vitro were X-gal positive. Of particular interest, bipotent erythroid megakaryocytic progenitors giving rise to mixed erythroid/megakaryocytic colonies were frequently encountered in yolk sac and fetal liver samples. Thus, by using this particular transgene, we uncovered in vivo a molecular pathway of differentiation that is active in early developmental stages but is less frequent in adult bone marrow samples.29 Sharing of certain transcription factors or of certain surface antigens between erythroid and megakaryocytic cells has been emphasized by Papayannopoulou et al29 and the sources cited for that study and could be the basis of activation of-globin in megakaryocytic cells. In view of
the high levels of LacZ expression in megakaryocytic cells, it
was of great interest to test whether endogenous murine -globin mRNA
could also be detected in positively selected CD41+ cells (FACS sorted
from in vitro Tpo-stimulated bone marrow cells), which included
megakaryocytic cells at different levels of development. Using
dilutions equivalent to approximately 13 CD41+ cells, we were able to
detect -globin mRNA by PCR (Asano H, unpublished data). Because CD41
is already expressed by a significant proportion of erythroid
progenitors (BFUe),29 it may be argued that the PCR results
stemmed only from the presence of erythroid progenitors in our samples.
However, no EKLF transcripts were detected in the same preparations,
making it unlikely that -globin in adult erythroid cells is
dissociated from EKLF activation.30 Rather, murine
-globin transcripts could be present in cells before their
definitive commitment to erythroid lineage or in nonerythroid-committed
(ie, Meg) cells and could provide added assurance that results
with LacZ in our mice had physiologic relevance and did not
result from the presence of the artificial construct. Although
-globin transcripts could be detected, no protein was detectable by
immunofluorescence. This suggests that in the absence of a proper
regimen of transcriptional factors, including EKLF, no efficient
translation or protein accumulation occurs.
Variegation in µLCR- pro
transgene were expected and were seen in our studies, as they were in
other studies using similar constructs.27, 28,31 For this
reason, only lines with high LacZ expression in red cells (80%
to 100% positive red cells) were selected for study. In addition to
variation in the proportion of positive cells, 3 additional features in
our transgenic animals were consistent with the graded rather than the
binary model of LCR-dependent position effects 27,32: (1)
the red cells of adult mice displayed significant variation in
intensity of staining, which was particularly evident when positivity
in cells was evaluated kinetically from 15 minutes to 2 hours; (2) the
proportion of FACS-gal(+)/TER119() (nonerythroid) cells was
higher in FL samples than in bone marrow-derived samples (Tables 1, 2)
(in the latter, the mean fluorescence intensity was also lower than in
fetal liver-derived cells [Figures 3, 4]); (3) the distribution of
FDG activity among positive cells was broad, both in fetal liver and
bone marrow-derived samples, consistent with varying levels of
LacZ among cells within a given sample.
In vivo activation of µLCR- promoter in
differentiated erythroid and megakaryocytic cells, as described in this
article, could be explained by 2 different hypotheses. First, the
activation can be at or after commitment to erythroid lineage, megakaryocytic lineage, or both. Second, the activation could
be before commitment to erythroid/nonmegakaryocytic lineage but
silenced later in other, nonerythroid/nonmegakaryocytic lineages. The
expectations from these 2 hypotheses are distinct. According to the
first hypothesis, which assumes expression of LacZ after commitment to erythroid/megakaryocytic lineage, if FACS-gal(+) progenitors exist, these should give rise only to erythroid colonies, megakaryocytic colonies, or both. By contrast, according to the second
hypothesis, which assumes expression of LacZ before commitment, positivity is expected in all types of committed progenitors. Results
of cultures of FDG(+) cells generating all types of progenitor cells,
especially CFU-GM, are highly compatible with the second hypothesis.
Indeed, the great majority of all progenitors was recovered in the
FDG(+) fraction. However, because both X-gal(+) BFUe-derived colonies
and X-gal(+) CFU-Meg-derived colonies were grown from FDG()
progenitors, the data further implied that activation of LCR-
promoter is not uniformly activated before erythroid commitment in all
progenitors and that it may not be occurring in a fraction of erythroid
or other progenitor cells. The distribution of scattered rather than
uniform X-gal cell positivity within erythroid bursts derived from
FDG() progenitors suggests that activation occurs after
commitment in a proportion of BFUe, as proposed in previous studies
using only X-gal staining in colonies from adult mice with low levels
of LacZ expression in red cells.28 Collectively,
our data suggest that although priming of lineage-restricted genes can
be detected in vivo in all types of progenitor cells, a spectrum of
molecular phenotypes is seen, indicating a gradual and progressive
final commitment to a specific lineage. Alternatively, one may suggest
that this low level of activation in progenitor cells is of a transient
nature alternating with states of no activation at any given time
("see-saw" phenomenon). Our data of LacZ activation in
µLCR-pro-LacZ mice share certain similarities with data in mice in which LacZ was linked to SCL promoter.34
Because SCL is a transcription factor essential for primitive and
definitive hemopoiesis, its activation in all types of progenitors was
not surprising. However, full activation was found only within
differentiated erythroid and megakaryocytic cells, similar to our findings.
We thank Dr H. Asano for performing the PCR in CD41+ cells and Gina
Alvino and Tyler Kimbrough for creating the µLCR-
Submitted February 9, 1999; accepted October 21, 1999.
Supported by National Institutes of Health grants DK30852 and HL46557.
Reprints: Thalia Papayannopoulou, Division of Hematology, University of Washington, Box 357710, Seattle, WA 98195-7710; email: thalp{at}u.washington.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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pro-LacZ as a
transgene
Top
Abstract
Introduction
-chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells.
Proc Natl Acad Sci U S A.
1992;89:3424
, -