By
From the * Department of Cellular and Molecular Biology, Immunology Group, S-223 62 Lund,
Sweden; and the Basel Institute for Immunology, CH-4005 Basel, Switzerland
Three lines of transgenic mice have been generated which express human CD25 under the
control of the 722-base pair region located immediately 5 of the precursor (pre)-B cell-specific
5 gene. All three strains express human CD25 in parallel to endogenous
5 on pre-B
cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human
CD25 on B lineage cells of transgenic mice has allowed the identification of a new
B220+CD19
5+ precursor of the B220+CD19+
5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19
precursors
have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration,
while the CD19+ pre-BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5
region of the
5 gene into the mouse genome confers tissue
and differentiation stage-specific expression of a transgene.
Two genes, VpreB and Differentially regulated gene expression has allowed to
define, and separate by FACS®, B lymphocyte lineage subpopulations in mouse bone marrow (BM). Hardy et al. (6)
have used the surface antigens heat stable antigen (HSA),
BP-1 and leukosialin (CD43) to separate B220 (CD45R)+
pre-B cell populations from immature IgM+ and mature
IgM+/IgD+ B cells. An analysis of the expression of VpreB,
Rolink et al. ordered B220+ B lineage subpopulations in
mouse BM by the analysis of the expression of c-kit, CD25,
and surrogate L chain (8) and by a single cell PCR analysis
of the IgH and L chain loci (9). Rolink et al. (10) then used
the differential expression of B220+ and CD19 to define a
B220+ CD19 In this paper we describe the generation of nine transgenic mouse lines in which the gene for the Construction of the Transgene Vector, 55, encode the proteins that together make up the surrogate L chain (1, 2). Both
genes are specifically expressed in precursor (pre)1-B cells,
but not in immature and mature, surface Ig-positive B cells,
nor in any other cell of mouse or human so far tested (for
review see reference 3). The pre-B cell-specific control of
5 gene expression has been analyzed in some detail (4, 5).
Deletion analysis of a 722-bp 5
region upstream of the
5
gene has defined two parts of the upstream region, A
5 and
B
5 (4). A
5, 154 bp directly 5
of the
5 gene, functions as
a basal promoter in different types of cells. B
5, 568 bp 5
of
A
5, acts in concert with A
5 as a suppressor region in nonpre-B cells, but as an enhancer region on a heterologous
promoter in pre-B cells (4, 5).
5, RAG-1 and RAG-2, TdT, mb-1 and bcl-2, and particularly the semiquantitation of DJH, VHDJH and V
J
rearranged Ig genes in populations of these B lineage cells have
allowed Li et al. (7) to propose an order of development of
the subpopulations. The earliest population, called fraction
A, which is CD43+, HSA
, BP-1
has low, if at all detectable, expression of VpreB,
5, RAG-1 and RAG-2, TdT,
and mb-1 and has low quantities of DJH rearranged IgH
chain genes while VH(J558)DJH and V
J
rearranged Ig genes
remained below detection limits.
cell population in BM, which could further
be subdivided into three subpopulations, a NK1.1+ precursor population of NK cells, a CD4+ population of unknown function, and a marker-negative population. These
cell populations are probably largely identical with Hardy's fraction A, since they were found to be CD43+, HSAlow,
BP-1
. The marker negative CD19
B220+ cells were suspected to contain some early B lineage progenitors, since
stromal cell/IL-7-reactive cells could be found in low frequencies (10).
chain of the
human IL-2 receptor (human CD25; hu-CD25) is under
the control of the 722-bp 5
region of the
5 gene (5
5).
In three of the lines the 5
5 confers lineage and differentiation stage specific-expression of the hu-CD25 transgene in
vivo.
5-hu-CD25 and Production
of Transgenic Mice.
5
5 (4) was made by PCR (PCR 1) using the
following primers: 5
primer: 5
ACGTCGACTTATATGTCACAGGCTGGCCTTGA 3
, 3
primer: 5
CATCAGCAGGTATGAATCCATTGACCCTCAAGTCCAAAGTC 3
. The hu-CD25 cDNA was made by PCR (PCR 2) using the following primers: 5
primer: 5
CTTGAGGGTCAATGGATTCATACCTGCTGATG 3
, 3
primer: 5
ACGGATCCCTAGATTGTTCTTCTACTCTTCCT 3
. The 5
5 3
primer and the
hu-CD25 5
primer contain overlapping sequences (underlined) to facilitate the third PCR. This (PCR 3) was made by using the 5
5 5
primer together with the hu-CD25 3
primer and as template use a 1:1 mixture of PCR 1 and PCR 2. The 5
5 5
primer
contained a SalI site and the hu-CD25 3
primer contained a
BamHI site and some extra bases for protection of the respective restriction site. The PCR product (PCR 3) was cloned into the vector pSPex23pA as a SalI/BamHI fragment. The pSPex23pA
vector contains human
-globin exon 2, intron 2, exon 3, and
polyA site (1.6-kb SalI/PstI fragment) from the vector pHSE3
(11) inserted into pSP73 (Promega Corp., Madison, WI) lacking
a BamHI site. The final 5
5-hu-CD25 vector contained in 5
to
3
order the following: 5
5 as promoter/enhancer, human CD25
cDNA, human
-globin exon 2, intron 2, exon 3, and polyA
site. This 3.2-kb SalI/XhoI fragment was purified and used for
injections.
-actin as
probes.
Antibodies, Flow Cytometric Analysis, and Cell Sorting. The rat mAb ACK-4 (anti-mouse c-kit; provided by Dr. S. Nishikawa, Kyoto University, Kyoto, Japan; reference 13) the rat anti-mouse IgD mAb NIM-R9; (provided by Dr. R. Parkhouse; reference 14) and the rat anti-mouse CD19 mAb 1D3 (10) were conjugated with biotin using standard procedures. The following rat mAbs were purchased from PharMingen (San Diego, CA) PE- and allophycocyanin-conjugated RA-6B2 (CD45R, B220), biotinylated 7D4 (anti-mouse CD25, TAC), PE-conjugated RM4-5 (anti- mouse CD4), PE-conjugated PK186 (anti-mouse NK1.1), and FITC- and PE-conjugated M-A251 (anti-human CD25). FITCconjugated goat anti-mouse IgM (µ chain specific) was obtained from Southern Biotechnology Associates (Birmingham, AL).
Single cell suspensions from BM were prepared by flushing out cells from femurs with either ice-cold staining buffer (PBS containing 2% FCS and 0.1% NaN3) or tissue culture medium (IMDM, 2% FCS). Cells were incubated with a combination of FITC-, PE-, biotin- or allophycocyanin-conjugated antibodies in staining buffer, washed with staining buffer, incubated for 15 min with PE-streptavidin (Southern Biotechnology Associates) to reveal the biotin reagent, and finally washed with staining buffer. Cells were analyzed on a FACScan® instrument (Becton Dickinson, Mountain View, CA). In double staining experiments propidium iodide was included in the staining buffer (1 µg/ml) to gate out dead cells. Cells present in the extended lymphocyte gate (low side scatter) were gated. Cell sorting was performed on a FACStar Plus® instrument. For the sorting of rare populations such as the B220+CD19Pre-B Cell Culture System.
Pre-B cell lines were established
from fetal livers of individual day 17 embryos from pregnant transgenic mice. The pre-B cell lines and clones were cultivated as described (15). In brief, pre-B cells were cultured on a semiconfluent layer of -irradiated (30 Gy) ST-2 stromal cells (16) in IMDM
containing 100 µg/ml kanamycin, 5 × 10
5 M 2-mercaptoethanol, 2% heat-inactivated FCS (GIBCO BRL, Gaithersburg, MD)
and 100 U/ml IL-7. Culture supernatant of J558L cells transfected with the murine IL-7 cDNA in the BMG neo vector was
used as a source of IL-7 (17). When stable pre-B cell lines were
established, the cells were transduced with a retrovirus containing
the mouse bcl-2 gene (a gift from Dr. M. Busslinger, Research
Institute of Molecular Pathology, Vienna, Austria) and transduced
cells were selected with 3 µg/ml puromycin on puromycin-resistant ST-2 cells. For the induction of differentiation of bcl-2 transfected pre-B cells, cells were harvested, washed three times in
medium without IL-7, and cultured on a semi-confluent layer of
-irradiated ST-2 stromal cells in medium without IL-7.
Limiting Dilution Analysis of Pre-B Cells Growth.
Cell suspensions from FACS® sorted cells were diluted by serial twofold dilutions in medium with IL-7 and were plated on a semi-confluent
layer of -irradiated (30 Gy) ST-2 cells in 96-well flat-bottom plates.
Cultures were scored on day 7 for pre-B cell colonies containing
>50 cells, using an inverted microscope. On several occasions,
the contents of individual wells with lymphoid colonies were removed and analyzed for B220 and CD19 expression by FACS®.
In all cases, the cells uniformly expressed B220 and CD19.
Northern Blot Analysis. Total cellular RNA was prepared using RNAzol B (Biotecx Laboratories, Inc., Houston, TX) according to the manufacturer's recommendations and analyzed as described (18). The complete cDNA of the hu-CD25 was used as a probe for the detection of hu-CD25 RNA.
Reverse Transcription-PCR.
RNA extraction, cDNA synthesis, and reverse transcription (RT)-PCR was done exactly as described (19). The following primer-pairs were used: hypoxanthine phosphoribosyl transferase (HPRT): 5 GCTGGTGAAAAG
GACCTCT 3
, 5
CACAGGACTAGAACACCTGC 3
;
5: 5
GAGATCTAGACTGCAAGTGAGGCTAGAG 3
, 5
CTTGGGCTGACCTAGGATTG 3
; hu-CD25/
-globin: 5 AGACCAGTCAGTTTCCAGGTGAA 3
, 5
AAGCGAGCTTAGTGATACTTGTG 3
. All PCRs were carried out with 1 cycle
94°C for 40 s, followed by 30 cycles at 94°C for 20 s, 55°C for 15 s,
and 72°C for 60 s. The probes specific for the HPRT,
5, and
hu-CD25 genes were generated by cloning PCR fragments into
pBluescript and were used for PCR-Southern blot analysis.
PCR Analysis of IgH Gene Rearrangements.
DJH rearrangements of
the H chain locus were amplified and detected using PCR as outlined in Fig. 4. Two forward primers binding immediately upstream of DFL/DSP elements or the DQ52 element, respectively,
were used in a mixture (DFS and DQ52 as described in reference
20) together with one reverse primer binding downstream of JH4
(JH4A in reference 16). In germline configuration, the DQ52 and
JH4A primers will amplify the 2.15-kb germline fragment. DJH1,
DJH2, DJH3, and DJH4 rearrangements involving either DFL, DSP,
or DQ52 elements will be detected by the emergence of bands of
1.46, 1.15, 0.73, and 0.20 kb, respectively.
Cells were sorted directly into 100 µl PBS containing 0.5% Tween 20 and proteinase K (0.2 mg/ml; Boehringer Mannheim GmbH, Mannheim, FRG) and incubated at 55°C for 2 h. The preparations were boiled and DNA was precipitated in ethanol with glycogen as the carrier. The DNA pellet was dissolved in Tris-HCl, pH 8.3. The DNA equivalent of 50 sorted cells was subsequently used for PCR amplification. The amplification protocol was 35 cycles of 20 s at 94°C, 30 s at 65°C, and 2 min at 72°C. An oligonucleotide specific for the JH4 region (5
The 722-bp region of the 5 upstream regulatory region
(4) was inserted upstream of the hu-CD25 cDNA (21) and
introduced as a transgene into the germline of mice. Cells
from BM and spleen of nine founder strains carrying the
transgene were analyzed by FACS® for hu-CD25 expression. Three of the nine strains were found to express surface hu-CD25. As determined by Southern and slot blot
analyses (Table 1), transgene copy numbers varied between 10 and 30 in expressing and between 4 and 400 in nonexpressing strains. This indicates that expression of hu-CD25
was most likely influenced by integration sites.
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The analysis of the cellularity as well as the staining of
the different B cell differentiation stages in BM and spleen
(as described in detail previously in reference 15) revealed
that in none of the transgenic lines was lymphocyte development disturbed by any means (Table 2) in agreement
with other studies using hu-CD25 as transgene (22). All
three founder strains expressing the transgene showed similar staining patterns in BM (as shown in Fig. 1 A for the
founder 82). Several levels (high, intermediate, and low) of
hu-CD25 expression were detected in B lineage cells in the
BM, while spleen and thymus cells were practically negative (Fig. 1 A). The ~0.5% of spleen cells which expressed
very low levels of hu-CD25 were found to be IgM+IgD
immature B cells (data not shown). Most hu-CD25 positive
BM cells expressed the B cell markers CD19 and CD45R
(B220; Fig. 1 A, and data not shown). RT-PCR analysis of
BM, spleen, liver, muscle, and heart readily detected huCD25 RNA in the BM and low levels in the spleen but no
signal was found in the nonlymphoid samples, while the
HPRT control primers detected HPRT RNA in all organs (Fig. 1 B). Thus, the 5
5 control region contains lineage- and differentiation stage-specific promoter/enhancer activity.
Three-color FACS® analysis of BM cells was performed
in order to define the stages at which the transgene was expressed and to analyze if this correlated with expression of
the endogenous 5 gene. Fig. 2 shows the analyses of the
expressor strains 82 and 83 in which cells expressing CD19
and a second marker were gated and the levels of hu-CD25
analyzed. Strain 73 showed virtually identical expression
levels of transgenic hu-CD25 to strain 82 (data not shown).
Strain 83 displayed ~5-10-fold lower levels when compared with the other two strains. While strains 82 and 73 both contained about 30 transgene copies, strain 83 contained about 10 copies. In BM cells from line 82 and 73, high
levels of hu-CD25 were found on c-kit+ pre-BI cells, intermediate levels were found on mouse CD25+ large pre-
BII cells, low expression on mouse CD25+ small pre-BII
cells, while very little or no expression was detected on immature (IgM+IgD
) and mature (IgM+IgD+) B lymphocytes. Expression of hu-CD25 protein correlated with transgenic RNA steady state levels were determined by RT-PCR
analysis; high levels of transgenic RNA were detected in
pre-BI cells, intermediate and low levels in large and small
pre-BII cells, respectively, and very little or no transgenic
RNA was detected in immature and mature B cells (not
shown). Parallel analysis of endogenous
5 gene transcript
levels demonstrated that
5 was expressed in a similar fashion, in agreement with previous analyses (19), with the exception that slightly lower relative amounts were detected in the later differentiation stages as compared to transgenic hu-CD25 RNA. Thus, in general, the expression of huCD25 protein and RNA correlated with that of endogenous
5. Both the endogenous
5 gene and the 5
5-controlled transgene generate transcripts which are expressed
in pre-BI cells and begin to be downregulated as these cells
differentiate into pre-BII cells.
The developmental control of transgenic hu-CD25 expression in B lymphocyte development was further analyzed in differentiating pre-B cells in vitro. c-kit+ pre-BI
cells can be expanded in vitro on stromal cells in the presence of IL-7 (15). Therefore, c-kit+ pre-BI cells from fetal
liver of transgenic mice were cultured on ST-2 stromal cells
in the presence of IL-7 and transduced with a retrovirus
encoding the bcl-2 cDNA to inhibit apoptosis after removal of IL-7. As shown in Fig. 3 A, the cells in culture
expressed CD19, 5 protein (as detected by the LM34 antibody; reference 23) and high levels of hu-CD25, but no
detectable IgM on the cell surface. Withdrawal of IL-7 and
analysis 1, 2, and 3 d thereafter demonstrated that the level
of CD19 expression remained constant while the surface
expression of
5 and hu-CD25 decreased with time. In addition, ~10% of the cells started to express IgM on the cell
surface at day 3 of differentiation. Furthermore, Northern blot analysis of these differentiating cells showed that the
RNA steady state levels of hu-CD25 and
5 decreased
with time of differentiation (Fig. 3 B). Disappearance of
hu-CD25 paralleled the disappearance of
5 RNA (Fig. 3
B). Again, these results indicate that the hu-CD25 transgene, like endogenous
5, is expressed in pre-BI cells. Expression of the endogenous
5 and hu-CD25 transgene is
downregulated as these pre-BI cells differentiate in vitro to
sIgM+ immature B cells.
Analysis of BM cells for hu-CD25 expression detected a
population of cells which did not express the B lineage
marker CD19, but did express intermediate levels of huCD25 (Figs. 1 and 4). This population was found to be
B220 positive (Fig. 4), and therefore, belongs to the mixture of B220+CD19 BM cells recently analyzed to be
composed of NK1.1+ precursors to NK, CD4+, and markernegative cells (10). B cell precursor activity was found only
in the marker-negative subpopulation. This marker-negative subpopulation amounts to approximately one-third of
all B220+CD19
cells, i.e., 1-2% of all BM nucleated cells
(10). In the 5
5-hu-CD25 transgenic mice the B220+
CD19
hu-CD25+ cells did not express NK1.1, or CD4
(Fig. 4), and are therefore found within the marker-negative
subpopulation. This marker-negative hu-CD25+ subpopulation comprises 10-15% of all B220+CD19
NK1.1
CD4
cells (Fig. 4), i.e., 0.1-0.3% of all BM cells.
The marker-negative subpopulation was sorted into huCD25+ and hu-CD25 cells and analyzed for other markers, in particular for the expression of endogenous
5. RTPCR analysis shown in Fig. 5 A revealed that these cells,
like CD19+ pre-BI cells coexpressed hu-CD25 and endogenous
5, whereas hu-CD25
sorted cells from the same
B220+CD19
population contained no detectable
5 or
hu-CD25 message. The B220+CD19+ hu-CD25+ cells
also expressed VpreB, sterile µ H chain, RAG-1, RAG-2, and B29 transcripts (data not shown). PCR analysis of the
H chain gene loci of the B220+CD19
hu-CD25+ cells revealed that a sizeable fraction of all loci were still in germline configuration while the rest of these loci were DJH rearranged (Fig. 5 B, lane 3). VHDJH rearranged loci could not be detected (data not shown). In CD19+ c-kit+ pre-BI
cells (lane 2) the H chain gene loci were found to be mostly
DJH rearranged in agreement with previous analyses (9).
We conclude from these analyses that the B220+CD19
NK1.1
CD4
hu-CD25+ cells contain early B lineage precursors. Since they have a considerable fraction of their
H chain loci in germline configuration they might be precursors of the CD19+ pre-BI cells. The B220+CD19
huCD25
5
BM cells might not belong to the B lineage.
These conclusions were further supported by an analysis of
the proliferation and differentiation potential of these early
B220+CD19
cell populations. Limiting dilution analyses
on stromal cells in the presence of IL-7 (15) showed that
the CD19+ hu-CD25high pre-BI cells (1 in 12) as well as
the B220+CD19
hu-CD25+ B lineage precursors (1 in
15) contained a similarly high frequency of clonable cells
(Fig. 5 C). This frequency of clonable cells in the B220+
CD19
NK1.1
CD4
hu-CD25+ cell population is, therefore, ~10-fold higher than the frequency found previously
by Rolink et al. (10) in the total B220+CD19
NK1.1
CD4
population. We therefore conclude that the huCD25 reporter transgene is preferentially expressed in early
clonable B lineage precursors before the expression of CD19.
It should be noted that in comparison to the pre-BI-
derived colonies, the B220+CD19 hu-CD25+-derived colonies were larger in size and contained more cells. After 7 d in
tissue culture, the CD19
hu-CD25+ precursors became
CD19 positive and the cells could be induced to differentiate
to IgM+ immature B cells upon removal of IL-7 from the cultures (data not shown). The B220+CD19
hu-CD25
cells
showed 20-30-fold lower frequency of clonable lymphoid cells in the above mentioned culture system (Fig. 5 C).
Collectively, these results show that the high expression of the transgenic hu-CD25 on the surface of BM cells has allowed the isolation and characterization of an early B lineage cell population that is likely to be precursors of pre-BI cells.
The three transgenic strains of mice presented here carry
the cDNA of the chain of the human IL-2 receptor
(CD25) under the control of the 722-bp 5
regulatory region of the
5 gene. The 5
5 regulatory region, consisting
of A
5 and B
5, conveys lineage and differentiation stage
specificity to the expression of the transgenic reporter gene,
hu-CD25. This expression pattern in vivo confirms and extends previous results from the analysis of various cell lines
representing different stages of B cell development (4, 5).
In the previous experiments, however, a heterologous enhancer was used in the reporter gene constructs in addition
to the 5
5 regulatory region to obtain measurable levels of
reporter gene expression. We show here that the short
stretch of the 722-bp 5
regulatory region contains the essential cis-acting regulatory elements for lineage and differentiation stage specific expression. Our experiments do not
provide any evidence for the need of additional enhancer elements for this specific expression. It will now be possible to
study the function of the 5
5 regulatory region in greater
detail.
It appears that the copy number of transgenes inserted
into the mouse genome is not important for the specificity
of expression. Furthermore, insertion of the transgene at
different sites in the genome only determines the level, and
not the specificity, of expression. These results indicate that
it will be possible to express other transgenes in a pre-B
cell-specific fashion without inserting them at the 5 locus
by homologous recombination.
Lineage- and tissue-specific expression conferred by a
short 5 region has also been reported for other genes.
Among the earliest genes analyzed in this context was the
rat insulin gene in which the 700-bp 5
region was shown
to drive the expression of the simian virus 40 large T antigen in
cells of the pancreas, and the transgenic mice developed
cell tumors (24). The insulin 5
region has subsequently been used to direct
cell-specific expression of
many genes. The insulin enhancer within these 700 bp, in
the context of its own promoter, confers gene expression only in
cells, while in the presence of a heterologous
promoter, the same enhancer is not as restricted and allows
expression in the brain also (25). Another example is the
620-bp proximal promoter of the lck gene. It was shown to
allow expression in thymocytes, but not in peripheral T cells,
in agreement with lck proximal promoter driven transcription in T cell development (26), though the defined differentiation stages in the thymus were not analyzed.
Perhaps the most widely used system expressing a transgene within the lymphoid system takes advantage of the well characterized IgH intron enhancer (Eµ) (27), either in combination with an Ig promoter or with a heterologous promoter. Such combinations allow expression not only in B, but also in T lymphocytes. Since Eµ seems to overrule the specificity of even a locus control region (30), it appears not suited for transgene expression if a more narrow window of expression is preferred.
In general, expression of the hu-CD25 transgene paralleled that of the endogenous 5 gene. In c-kit+ pre-BI
cells and in large pre-BII cells,
5 and transgene expression was highest. In these differentiation stages, using currently available reagents,
5 protein is detectable either in the cytoplasm (8) or on the cell surface (31). Small differences can
be seen in the small pre-BII/immature B cell compartments. The data shown in Fig. 2 indicate that the huCD25 is expressed as protein in small pre-BII cells, and at
even lower levels in immature B cells in two of the three
founder lines. Endogenous
5 protein was not detectable at
the later differentiation stages and
5 mRNA was reduced
about 20-30-fold when compared to pre-BI cells (reference 19 and unpublished observation). The hu-CD25 transgene contains human
-globin sequences which might prolong mRNA half-life as compared to endogenous
5 and,
hence, protein synthesis. In addition, hu-CD25 protein might
be more resistant than
5 to degradation which again could
contribute to prolonged expression of hu-CD25 in differentiating B lineage cells in BM.
The easily detectable expression of the transgenic huCD25 protein on the surface of cells has permitted the
identification of a CD19B220+ hu-CD25+ cell population in the BM that are NK1.1
and CD4
. These novel
cells were also found to express endogenous
5, and as a
population, to contain cells that have started to rearrange their IgH gene loci. A high proportion of these CD19
B220+ hu-CD25+ cells are clonable on stromal cells in the
presence of IL-7. In fact, the frequency of clonable cells is
as high as in the CD19+B220+ hu-CD25high c-kit+ pre-BI
cell population. Given that the plating efficiency in these
cultures is not 100% (32), the actual frequencies may be
even higher. At present it can not be completely ruled out, however, that the CD19
B220+ hu-CD25+ cell population, which represents about 5% of all CD19
B220+ in the
BM of transgenic mice, is nevertheless heterogeneous. The
finding that the CD19
hu-CD25+ population contains
much more of the IgH loci in germline configuration argues that the CD19
hu-CD25+ cells are the precursors of
the CD19+ pre-BI cells. Our results agree with previous
analyses of the configuration of IgH and L chain loci undertaken by Li et al. (7) for Hardy's fraction A of mouse
BM, and suggest that the DJH rearranged IgH chain loci
found by these authors are contributed by B220+CD19
NK1.1
CD4
cells, now further marked by the hu-CD25
reporter transgene.
To date, it is clear that cells expressing CD19 are committed to the B cell lineage. However, stages of hemopoiesis before B cell commitment are poorly understood. The hu-CD25 transgenic mice described in this paper may help to further characterize these early stages of B lineage development and commitment. In addition, they may help further in elucidating the early arrests in B cell development observed in mutant mice defective for, e.g., E2A (33, 34) or EBF (35).
Address correspondence to Fritz Melchers, Basel Institute for Immunology, Grenzacherstr. 487, CH-4005 Basel, Switzerland. T.H. Winkler's present address is Section of Immunology, Medical Department III, University of Erlangen-Nurnberg, Schwabachanlage 10, D-91054 Erlangen, Germany.
Received for publication 9 September 1996
1 Abbreviations used in this paper: BM, bone marrow; HPRT, hypoxanthine phosphoribosyl transferase; HSA, heat stable antigen; hu-CD25, human CD25; pre, precursor; RT, reverse transcription.We thank U. Müller and A. Werner for excellent technical assistance, M. Dessing for skillful cell sorting, and Drs. A. Rolink, R. Ceredig, K. Karjalainen, and H.-R. Rodewald for discussions and critically reading our manuscript.
This work was supported in part by the Swedish Medical Research Council, the Medical Faculty Lund, the Kungliga Fysiografiska Society, the Österlunds, the Kocks, the Crafoords, and the G. Danielssons Foundations (I.-L. Mårtensson). The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd. (Basel, Switzerland).
1. | Sakaguchi, N., and F. Melchers. 1986. Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature (Lond.). 324: 579-582 [Medline] . |
2. | Kudo, A., and F. Melchers. 1987. A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes. EMBO (Eur. Mol. Biol. Organ.) J. 6: 2267-2272 [Abstract] . |
3. | Melchers, F., H. Karasuyama, D. Haasner, S. Bauer, A. Kudo, N. Sakaguchi, B. Jameson, and A. Rolink. 1993. The surrogate light chain in B-cell development. Immunol. Today 14: 60-68 [Medline] . |
4. |
Martensson, I.-L., and
F. Melchers.
1994.
Pre-B cell specific
![]() |
5. | Yang, J., M.A. Glozak, and B.B. Blomberg. 1995. Identification and localization of a developmentally stage-specific promoter activity from the murine lambda 5 gene. J. Immunol. 155: 2498-2514 [Abstract] . |
6. | Hardy, R.R., C.E. Carmack, S.A. Shinton, J.D. Kemp, and K. Hayakawa. 1991. Resolution and characterization of proB and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173: 1213-1225 [Abstract] . |
7. | Li, Y.S., K. Hayakawa, and R.R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178: 951-960 [Abstract] . |
8. |
Rolink, A.,
U. Grawunder,
T.H. Winkler,
H. Karasuyama, and
F. Melchers.
1994.
IL-2 receptor ![]() |
9. | ten Boekel, E., F. Melchers, and A. Rolink. 1995. The status of Ig loci rearrangements in single cells from different stages of B cell development. Int. Immunol. 7: 1013-1019 [Abstract] . |
10. | Rolink, A., E. ten Boekel, F. Melchers, D.T. Fearon, I. Krop, and J. Andersson. 1996. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J. Exp. Med. 183: 187-194 [Abstract] . |
11. |
Pircher, H.,
T.W. Mak,
R. Lang,
W. Ballhausen,
E. Rüedi,
H. Hengartner,
R.M. Zinkernagel, and
K. Bürki.
1989.
T
cell tolerance to Mlsa encoded antigens in T cell receptor
V![]() |
12. | Bucchini, D., C.A. Reynaud, M.A. Ripoche, H. Grimal, J. Jami, and J.C. Weill. 1987. Rearrangement of a chicken immunoglobulin gene occurs in the lymphoid lineage of transgenic mice. Nature (Lond.). 326: 409-411 [Medline] . |
13. | Ogawa, M., Y. Matsuzaki, S. Nishikawa, S.I. Hayashi, T. Kunisada, T. Sudo, T. Kina, H. Nakauchi, and S.I. Nishikawa. 1991. Expression and function of c-kit in hemopoietic precursor cells. J. Exp. Med. 174: 63-71 [Abstract] . |
14. |
Parkhouse, R.M.E.,
G. Preece,
R. Sutton,
J.L. Cordell, and
D.Y. Mason.
1992.
Relative expression of surface IgM, IgD
and the Ig-associating ![]() ![]() |
15. | Rolink, A., A. Kudo, H. Karasuyama, Y. Kikuchi, and F. Melchers. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO (Eur. Mol. Biol. Organ.) J. 10: 327-336 [Abstract] . |
16. | Ogawa, M., S. Nishikawa, K. Ikuta, F. Yamamura, M. Naito, K. Takahashi, and S.I. Nishikawa. 1988. B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST-2: B cell progenitor develops first in the embryonal body rather than in the yolk sac. EMBO (Eur. Mol. Biol. Organ.) J. 7: 1337-1343 [Abstract] . |
17. | Karasuyama, H., and F. Melchers. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using a modified cDNA expression vectors. Eur. J. Immunol. 18: 97-104 [Medline] . |
18. |
Grawunder, U.,
F. Melchers, and
A. Rolink.
1993.
Interferon-![]() |
19. | Grawunder, U., T.M.J. Leu, D.G. Schatz, A. Werner, A.G. Rolink, F. Melchers, and T.H. Winkler. 1995. Downregulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement. Immunity. 3: 601-608 [Medline] . |
20. | Ehlich, A., V. Martin, W. Müller, and K. Rajewsky. 1994. Analysis of the B-cell progenitor compartment at the level of single cells. Curr. Biol. 4: 573-593 [Medline] . |
21. | Leonard, W.J., J.M. Depper, G.R. Crabtree, S. Rudikoff, J. Pumphrey, R.J. Robb, M. Krönke, P.B. Svetlik, N.J. Peffer, T.A. Waldmann, and W.C. Greene. 1984. Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature (Lond.). 311: 626-631 [Medline] . |
22. | Nishi, M., Y. Ishida, and T. Honjo. 1988. Expression of functional interleukin-2 receptors in human light chain/Tac transgenic mice. Nature (Lond.). 331: 267-269 [Medline] . |
23. | Karasuyama, H., A. Rolink, and F. Melchers. 1993. A complex of glycoproteins is associated with VpreB/lambda 5 surrogate light chain on the surface of µ heavy chain-negative early precursor B cell lines. J. Exp. Med. 178: 469-478 [Abstract] . |
24. |
Hanahan, D..
1985.
Heritable formation of pancreatic ![]() |
25. | Dandoy-Dron, F., J.-M. Itier, E. Monthioux, D. Bucchini, and J. Jami. 1995. Tissue-specific expression of a rat insulin 1 gene in vivo requires both the enhancer and the promotor regions. Differentiation. 58: 291-295 [Medline] . |
26. | Allen, J.M., K.A. Forbush, and R.M. Perlmutter. 1992. Functional dissection of the lck promotor. Mol. Cell Biol. 12: 2758-2768 [Abstract] . |
27. | Neuberger, M.S.. 1983. Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. EMBO (Eur. Mol. Biol. Organ.) J. 2: 1373-1378 [Medline] . |
28. | Gillies, S.D., S.L. Morrison, V.T. Oi, and S. Tonegawa. 1983. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell. 33: 717-728 [Medline] . |
29. | Banerji, J., L. Olson, and W. Schaffner. 1983. A lymphocytespecific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 33: 729-740 [Medline] . |
30. | Elliot, J.I., R. Festenstein, M. Tolaini, and D. Kioussis. 1995. Random activation of a transgene under the control of a hybrid hCD2 locus control region/Ig enhancer regulatory element. EMBO (Eur. Mol. Biol. Organ.) J. 14: 575-584 [Abstract] . |
31. | Winkler, T.H., A. Rolink, F. Melchers, and H. Karasuyama. 1995. Precursor B cells of mouse bone marrow express two different complexes with the surrogate light chain on the surface. Eur. J. Immunol. 25: 446-450 [Medline] . |
32. | Rolink, A., D. Haasner, S. Nishikawa, and F. Melchers. 1993. Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice. Blood. 81: 2290-2300 [Abstract] . |
33. | Bain, G., E.C. Maandag, D.J. Izon, D. Amsen, A.M. Kruisbeek, B.C. Weintraub, I. Krop, M.S. Schlissel, A.J. Feeney, M. van-Roon, et al . 1994. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell. 79: 885-892 [Medline] . |
34. | Zhuang, Y., P. Soriano, and H. Weintraub. 1994. The helixloop-helix gene E2A is required for B cell formation. Cell. 79: 875-884 [Medline] . |
35. | Lin, H., and R. Grosschedl. 1995. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature (Lond.). 376: 263-267 [Medline] . |