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INTRODUCTION |
CD45 is a high molecular weight, transmembrane protein-tyrosine
phosphatase expressed on all nucleated cells of hematopoietic origin.
The protein is structurally heterogenous, consisting of isoforms
ranging in size from 180 to 220 kDa. The heterogeneity of the
protein results from differential RNA splicing of at least five and
perhaps six exons encoding part of the extracellular domain (1-3). The
CD45 exon usage pattern is highly regulated by the different leukocyte
populations and is cell type-specific. The predominant isoform
expressed by B cells contains all variable exons (B220+)
(4, 5). Upon antigen stimulation, lower molecular weight isoforms are
detected (6, 7). Several subtypes of antigen-specific memory B cells
have been identified, including an antibody-secreting subset producing
the full-length splice variant and a nonsecreting subtype, which is
B220
(8). In thymocytes three or four variable exons are
removed to generate the lowest molecular weight isoforms (1, 3, 4,
9-11). Peripheral T cells express multiple splice variants and have a
varied isoform expression pattern that is dependent upon the
differentiation state, function, and prior antigenic exposure (2). Mast
cells and monocytes also produce specific sets of CD45 isoforms, which
are distinctive for each myleoid cell type (12).
In addition to the protein structural differences, polymorphic sequence
variations in the CD45 gene led to the identification of three alleles
in inbred murine strains, Ly5a (CD45.1),
Ly5b (CD45.2), and Ly5c (13). The
CD45.2 allele is expressed by most of the established strains, while
the CD45.1 allele is found in only a few (13). The CD45.1 and CD45.2
alleles are distinguished antigenically by their reactivity to specific
monoclonal antibodies, and the nucleotide changes that produce the
antigenic differences have been identified (3).
CD45 is encoded by a single gene, and expression appears to be
regulated at the level of transcription (14). The murine gene is
characterized by 34 exons and a large 50-kilobase
(kb)1 intron between exons 2 and 3. CD45 transcription can initiate at three distinct sites in exon
1a, exon 1b, and downstream of exon 1b (15). However, the sequences
responsible for the developmental and tissue-specific transcription of
CD45 have yet to be identified.
Extensive analyses have demonstrated that CD45 is required to generate
signaling through both the T and B cell receptors (16-21). In the case
of antigen receptor signaling, CD45 is required for both the positive
and negative regulation of the Src family kinases associated with the
antigen receptors (22-25). Mice in which the expression of CD45 is
impaired (26) or absent (27, 28) have greatly reduced numbers of
peripheral T cells, suggesting that CD45 is required for T cell
development (26, 27). In contrast to T cell maturation, there appears
to be little effect on B cell development in the null mice (26, 27).
The role of CD45 in signaling in other lymphoid lineages is less well
defined, although reports suggest that the CD45 phosphatase activity is
important in regulating signal transduction in mast cells through the
high affinity receptor for IgE, Fc
RI (29, 30).
Much less is known about the functional significance of the
extracellular domain, although the isoform differences most likely convey ligand specificity. Studies have shown that the isoforms differentially affect activation through the T cell receptor (31, 32)
and that the isoforms vary in their associations with the receptor (33,
34). The expression of specific isoforms has also been implicated in T
cell apopotosis (35-37).
Many of the CD45 expression experiments were conducted with cDNA
transgenes flanked either by retroviral sequences (38) or by the
thymocyte-specific proximal lck promoter and the human growth hormone minigene (39, 40). Expression from these CD45 cDNA
constructs is not obtained consistently (41), and in transgenic mice,
the lck-controlled cDNAs only express 10-30% of
endogenous CD45 protein levels (40). The 5' and 3' sequences used to
regulate expression from these cDNA constructs differ considerably
from those controlling the endogenous CD45 gene. The proximal
lck promoter is expressed in thymocytes but not in
peripheral T cells (42), a pattern of expression that is much
restricted compared with endogenous CD45. In contrast, expression from
the retroviral LTR promoter should be constitutive, but this construct
lacks a discernible polyadenylation sequence in proximity to the CD45
cDNA. In addition, the 3' sequences may contribute significantly to
the expression of these cDNAs. Signals for controlling mRNA
translation, stability, and localization have been found in the 3'-UTR
of some genes, indicating that these sequences can contain key
information for both positive and negative regulation of mRNA (43,
44). Therefore, the difficulty in consistently obtaining expression
from these CD45 cDNA constructs may be due in part to the 5' and 3'
regulatory elements used.
To address the roles of these gene elements, we designed new CD45
cDNA vectors. Sections of the 3'-end of the CD45 gene downstream of
the translational termination codon were evaluated in reporter gene
constructs and in CD45 cDNA vectors. Regions of the 5'-end of the
CD45 gene as large as 19 kb upstream of the initiation codon were
tested for promoter activity and compared with the promoter for the
human LFA-1 gene, a gene with similar expression characteristics to
CD45. We also tested the effect of the introns between exons 3 and 9 in
CD45 expression constructs. These introns flank the principal
alternatively expressed exons 4-8. Here we report that by using these
novel constructs, we are able to identify components that generate
reproducible expression of CD45 in a variety of hematopoietic cell
types. These results suggest that the CD45 intron sequences contain
information necessary for expression from the transgenes. Furthermore,
the intron sequences flanking the alternatively spliced exons provide
correct leukocyte lineage-specific splicing.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Transfection--
70Z/3 is a
methylnitrosourea-induced murine pre-B lymphoblast cell line.
Transfection of 70Z/3 was performed by electroporation in cuvettes with
a 3.5-mm gap. After adding 25 µg of linearized DNA to 1 × 107 cells in phosphate-buffered saline (PBS), the DNA-cell
complex was kept on ice for 10 min and then placed in a BTX Transfector 300 (BTX, La Jolla, CA) for electroporation (settings: capacitance, 1400 microfarads; voltage, 200 V). Following electroporation, the
samples were incubated on ice for 10 min and then resuspended in growth
medium for 48 h. Cells were subsequently grown in medium containing 1.5 mg/ml G418 sulfate (G418; Omega Scientific,
Tarzana, CA).
MD.45-27J (27J) is a cytotoxic murine T cell hybridoma clone obtained
from Joseph Lustgarten (Sidney Kimmel Cancer Center, La Jolla, CA)
(45). Twenty micrograms of linearized plasmid DNA were mixed with
1 × 107 cells in PBS, and immediately electroporation
(800 microfarads and 250 V) was performed. After electroporation, the
cells were placed on ice for 10 min and then resuspended in growth
medium for 36 h. Cells expressing the neomycin gene were selected
using 2.0 mg/ml G418.
BW5147, a murine thymic lymphoma cell line, was transfected using
1 × 107 cells in PBS and 25 µg of linearized
plasmid DNA. The mixture of cells and DNA was incubated on ice for 10 min prior to electroporation (250 V and 2200 microfarads). After
electroporation, the cells were placed on ice for 10 min and then
resuspended in growth medium. After 48 h, the cells were placed in
medium containing 1.0 mg/ml G418.
Human embryonal kidney cells (293) were transfected using LipofectAMINE
Reagent (Life Technologies, Inc.). One day before transfection,
1.2 × 106 cells were plated on 10-cm dishes treated
with 0.1 mg/ml poly-D-lysine (Sigma). For transfection, 6 µg of supercoiled plasmid DNA, 40 µl of LipofectAMINE, and 300 µl
of unsupplemented growth medium were mixed and kept for 15 min. at room
temperature. After the 293 cells were washed two times with
unsupplemented medium, the DNA/lipid mixture was added and incubated
with the cells for 5 h at 37 °C. The cells were washed twice
with supplemented medium and grown in 10 ml of medium for 48 h.
S49 is a murine T lymphoma cell line provided by Robert Hyman (The Salk
Institute, La Jolla, CA). Transfection was performed by electroporation
(250 V, 1400 microfarads). The human T cell leukemia line, Jurkat, was
obtained from Javi Piedrafita (Sidney Kimmel Cancer Center, La Jolla,
CA). Jurkat was transfected using DMRIE-C reagent (Life Technologies)
according to the manufacturer's directions.
All cell lines were obtained from ATCC (Manassas, VA) unless otherwise
indicated. The growth medium for the cell lines was Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum, 10 units/ml penicillin, and 10 µg/ml streptomycin except for 70Z/3,
which was grown in RPMI 1640 medium supplemented with 2 mM
L-glutamine, 10 mM HEPES, 1.0 mM
sodium pyruvate, 10% fetal bovine serum, 0.05 mM
2-mercaptoetanol, 10 units/ml penicillin, and 10 µg/ml streptomycin.
All media components were from Irvine Scientific (Irvine, CA).
Plasmids and Vector Constructions--
To study CD45 cDNA
expression in a variety of leukocyte populations, we obtained three
constructs. The plasmid, pARV/CD45 (H), was kindly provided by K. Bottomly (Yale University School of Medicine, New Haven, CT). This
plasmid is a modification of pARV/CD45 (38) in which the neomycin gene
was replaced with the hygromycin gene (31). Since selection for
hygromycin resistance in leukocytes is inefficient, we removed the
hygromycin gene as a ClaI fragment and reinserted the
neomycin gene (GKneo), creating pARV/CD45 (N). The neomycin gene from
pGKneocbpA (Eva Lee, University of California, San Diego,
La Jolla, CA) was used and contains the mammalian phosphoglycerate
kinase promoter and the bovine growth hormone poly(A) sequence. The
CD45 cDNA plasmids, pML84 and pML171, were obtained from J. Marth
(Department of Medicine and Division of Cellular and Molecular
Medicine, University of California, San Diego, La Jolla, CA). The
full-length cDNA is present in pML84 (CD45RABC), while
pML171 carries the CD45 cDNA encoding the isoform missing exons
4-6 (CD45RO). In these vectors, the transgenes are
inserted 3' of the proximal lck promoter and upstream of the
human growth hormone gene (40). We added the GKneo NotI
fragment into the NotI site of each plasmid to facilitate
selection of stable transfectants. In all of the cDNA transgenes,
the 3200-base pair (bp) XbaI fragment containing the CD45.2
allelic sequence was replaced with the corresponding XbaI
fragment specifying the sequences for the CD45.1 allele.
To test the 3' CD45 sequences, the plasmids pSPORT-(
A), pSPORT-BGA,
pSPORT-XR, and pSPORT-EA, were generated (Fig. 1A). These plasmids are derivatives of pCMVSPORT
gal (Life Technologies). The
SV40 small t/poly(A) site of pCMVSPORT
gal was removed creating pSPORT-(
A) (Fig. 1A). The SV40 small t/poly(A) was
replaced by a 280-bp XbaI to XhoI bovine growth
hormone poly(A) insert from pGKneocbpA to produce
pSPORT-BGA (Fig. 1A). An 868-bp XbaI to
EcoRI piece from CD45 exon 33 was introduced into
pSPORT-(
A), generating pSPORT-X (not shown). The XbaI site
lies 15 bp downstream from the CD45 stop signal, and the
EcoRI site is 165 bp upstream from the 3'-end of the CD45
mRNA (3). An adjacent 800-bp EcoRI fragment containing
the remaining 3' portion of exon 33 and all CD45 polyadenylation signals were cloned into pSPORT-X, resulting in pSPORT-XR (Fig. 1A). This EcoRI fragment also was introduced into
pCMVSPORT
gal upstream from the SV40 small t/poly(A) creating
pSPORT-EA (Fig. 1A).

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Fig. 1.
Structure of the plasmid constructs.
CD45 3' sequences are represented by diagonal
striped boxes. CMV, the
cytomegalovirus promoter; LFA, the human LFA-1 (CD11a)
promoter. A, all plasmids of the -galactosidase
expression constructs were derived from pCMVSPORT gal shown at the
top. The components of the plasmids are -galactosidase
(filled box), SV40 small t/poly(A)
(open box), and bovine growth hormone poly(A)
(vertical striped box). The CD45
element in pSPORT-EA lacks the XbaI to EcoRI
3'-UTR sequence present in pSPORT-XR. pSPORT-EA retains the 3'-terminal
165 bp of the 3'-UTR and the following 750 bp of downstream sequence
(diagonal striped box). The
polyadenylation site in the CD45 3' sequence is indicated
(pA). B, the structure of two CD45 cDNA
plasmids is shown with restriction sites used for the construction in
the map at the top. The positions of the translation start
and stop codons are indicated. The CD45 cDNA region extending from
exon 1b through the XbaI site of exon 33 is designated
1b-33. C, the composition of the CD45 minigene
and partial restriction map of the construct are shown. The CD45 5'
cDNA region that contains exons 1b through 3 is indicated as
1b-3. The genomic DNA encoding the alternative spliced exons
4-8 is shown by the solid line with the exons
indicated. The 3' cDNA sequences are designated 9-33. Otherwise,
the components are the same as in pLFAT200 (B).
D, the structures of CD45 minigene constructs under the
control of various extensions of CD45 upstream sequences and a partial
restriction map are shown. The SfiI site is not a naturally
occurring site as discussed under "Experimental Procedures." The
upstream region is indicated with the solid line
with the relevant exons shown. The NheI site lies within
exon 1b. The remainder of the iT200 minigene extends 3' from this
NheI site and is shown by the dashed
line.
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To study the effect of 5' sequences on CD45 cDNA expression, the
constructs pLFAT200 and pCMVT200 (Fig. 1B) were made by
subcloning all components into pBluescript KS (Stratagene, La Jolla,
CA). The 5' cDNA from T200/3/pAX (38) containing all alternatively spliced exons (exons 4-8) was inserted as a
ClaI/XbaI fragment. The 3' CD45 DNA was subcloned
from pSPORT-XR as a 1800-bp XbaI/EcoRI fragment
(striped box in Fig. 1A). The
remaining cDNA region obtained from a CD45.1 allele (3) extended
from exon 9 through 33 and was inserted as a 3200-bp XbaI
fragment. A 1800-bp SalI/ClaI fragment containing
the human leukocyte function-associated antigen (LFA-1, CD11a) promoter
was inserted 5' of the CD45 cDNA sequences. The LFA-1 promoter was
obtained by PCR amplification from DNA isolated from the human T
lymphocyte line, Jurkat. The sense primer extended from position
1694
to
1668 of the LFA-1 promoter (46) and contained unique
SalI and SfiI sites at the 5'-end. The antisense primer extended from +77 to +96 of the LFA-1 promoter (46) and had
unique ClaI and SgfI sites engineered at the
3'-end of the LFA-1 sequence. The construct, pLFAT200 (Fig.
1B), was completed by the addition of the neomycin gene
(GKneo). The LFA promoter was replaced with the cytomegalovirus (CMV)
promoter to generate pCMVT200 (Fig. 1B) by digestion of
pLFAT200 with SfiI and ClaI and insertion of a
NruI/HindIII fragment from pcDNA3
(Invitrogen, Carlsbad, CA).
To investigate regulatory sequences within the introns, the CD45
minigene construct, pLFAiT200 (Fig. 1C), was made by
inserting all components into a modified mammalian expression vector
pcDNA3 in which the CMV promoter had been replaced by the 1800-bp
fragment containing the LFA-1 promoter. The 5' CD45 cDNA/intron
region in LFAiT200 starts in exon 1b at an NheI site, 27 bp
from the beginning of the exon, continues through exons 2 and 3, and
ends within the 3/4 intron at a BglII site, 500 bp
downstream from exon 3. Two PCR products were used to generate this
region. PCR amplification of cDNA extending from exon 1b to 3 produced the first fragment, and amplification of CD45 genomic DNA
containing exon 3 and intron sequences 3' to exon 3 resulted in the
second. The fragment was completed by ligating the two PCR products. A
genomic clone carrying CD45 exons 3-8 was used to obtain a
BglII to BamHI fragment containing exon 4. This
fragment was inserted downstream from exon 3 at the BglII
(intron) and BamHI (vector) sites. The genomic sequences,
extending from the BamHI site 550 bp upstream of exon 5 to
an XhoI site 600 bp downstream of exon 8, were taken from a
genomic clone, placed into the plasmid vector pGEM11Z (Promega,
Madison, WI) and subsequently transferred to the minigene construct as
a BamHI/NotI fragment generating plasmid
pLFA1-8. The genomic region containing exons 8 and 9 was amplified by
PCR from mouse genomic DNA and subcloned as a 2000-bp HindIII/EcoRI fragment into pGEM11Z, creating
pG11(8-9). For this amplification, the 35-bp sense oligonucleotide
primer was the complete exon 8 sequence, and the antisense primer
extended from base 192 to 215 of exon 9. The PCR amplification fragment
included the XhoI site downstream from exon 8 and the
XbaI restriction site within exon 9. The XbaI to
EcoRI 3' CD45 DNA fragment in pSPORT-XR was inserted into
pG11(8-9) as an XbaI/NotI fragment generating
pG11(8-9)A. A CD45.1 cDNA region (3) extending from exon 9 to 33 was subcloned into pG11(8-9)A as an XbaI fragment producing
pG11(8-A). The CD45 minigene construct, pLFAiT200, was completed by the
introduction of the XhoI/NotI restriction
fragment from pG11(8-A) into pLFA1-8. The final construct contains
CD45 sequence consisting of a cDNA 5' region (exons 1b-3), a
genomic DNA region extending from exon 3 to exon 9, a cDNA segment
from exon 9 to exon 33, and 3' gene sequences from exon 33 to 750 bp downstream of the end of transcriptional termination.
The potential promoter region upstream of exon 1a was isolated from a
genomic library constructed in the LambdaGEM-12 cloning vector
(Promega). The longest
clone extended ~19 kb upstream and 1800 bp
downstream of the CD45 translational start site. The insert was flanked
by NotI sites from the vector. A partial restriction map of
a subsection of this DNA is shown in Fig. 1D; the
NheI site maps within exon 1b. The entire upstream region
was subcloned into plasmid pGEM5Z as three smaller fragments: a 15-kb
NotI/PstI fragment consisting of the most
upstream 5' sequences (pG5NP); a 1.3-kb PstI fragment
(pG5P); and a 4.5 kb, PstI/NotI fragment including exons 1a, 1b, and 2 as well as 1.7 kb of intron sequence 3'
of exon 2 (pG5PN). The 5' NotI site was changed to a
SfiI site using double-stranded oligonucleotides containing
a NotI overhang and an internal SfiI site. Using
the natural EcoRI, PstI, and NheI
sites and the synthetic SfiI site, the following plasmids were generated: pCD45FiT200, pCD45EiT200, pCD45PiT200, and
pCD45P2iT200 (Fig. 1D).
RNA Isolation--
Total RNA was purified from cells using
TRIZOL (Life Technologies) following the manufacturer's directions.
Fifty micrograms RNA isolated from cells transfected with plasmids
pLFAT200 and pCMVT200 were treated with 1 unit of DNase I (Promega) for
1 h at 37 °C. The DNase I was removed by one extraction with
phenol/chloroform/isoamyl alcohol followed by one extraction with
chloroform/isoamyl alcohol as suggested by Promega. The RNA was
precipitated with ethanol and resuspended in RNase-free water.
Reverse Transcription and Polymerase Chain Reaction
(RT-PCR)--
RT-PCR was performed using the GeneAmp RNA PCR kit
(Applied Biosystems, Foster City, CA) to distinguish transgene CD45.1
mRNA transcripts from the endogenous CD45.2 transcripts of the
transfected cells. All RT and PCR reactions were assembled according to
the manufacturer's recommendations. The antisense oligonucleotide used
for cDNA synthesis, 5'-GAGACCAGAAACTCATAG-3', contains the last 13 bp of exon 14 and the first 5 bp of exon 15. For the PCR component, the
sense oligonucleotide primer was the complete exon 3 sequence,
5'-GGCAAACACCTACACCCAGTGATG-3'. The antisense oligonucleotides are
specific for the CD45.1 (Ly5a) and CD45.2
(Ly5b) alleles (3, 47) and consist of bases
86-106 of exon 12. The sequences of these antisense primers are as
follows with the allele-specific bases underlined: CD45.1,
5'-CCATGGGGTTTAGATGCAGGA-3' and CD45.2,
5'-CCATGGGGTTTAGATGCAGAC-3'. The cDNA was synthesized for 1 h at 42 °C followed by 5 min at 95 °C. The cDNA
was amplified for 34 cycles. The first cycle consisted of 2 min at
94 °C, 1 min at 63 °C, and 3 min at 72 °C. The remaining
cycles were 1 min at 94 °C, 1 min at 63 °C, and 3 min at
72 °C. The amplified products were separated on a standard
TBE-agarose gel (48), photographed using a DC120 zoom digital camera
(Eastman Kodak Co.), and analyzed using Kodak 1D imaging analysis software.
-Galactosidase Assays--
293 cells were harvested 48 h
after transfection. The plates were washed twice in PBS and then
incubated with 2 ml of 0.5 g/liter trypsin, 0.2 g/liter EDTA solution
(Irivine Scientific) for 5 min at 37 °C. The isolated cells were
washed twice in PBS and resuspended in 1 ml of ice-cold PBS. Cellular
extracts were prepared by three cycles of freeze-thaw followed by
removal of the cellular debris and assayed for
-galactosidase
activity (48). Total protein in the extracts was measured using the
Bio-Rad Protein Assay (Bio-Rad).
Flow Cytometry and Antibodies--
Cell suspensions were stained
using directly conjugated antibodies from Pharmingen (La Jolla, CA):
FITC-anti-CD45.1, FITC-anti-CD45.2, and FITC-anti-THP
(IgG2a isotype control). Approximately 1 × 106 cells were resuspended in 100 µl of Hanks' balanced
salt solution (Mediatech, Herndon, VA) containing 0.1% bovine serum
albumin and 0.02% sodium azide. Antibodies were added to a final
concentration of 0.5 µg/ml and incubated in the dark for 20 min at
4 °C. Cells were washed one time in Hanks' balanced salt solution,
resuspended in 500 µl of Hanks' balanced salt solution, and analyzed
by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA)
using CellQuest software. Live cells were discriminated from dead cells using propidium iodide at a final concentration of 5 µg/ml.
Sequencing--
Correct CD45 cDNA sequences in the
constructs were confirmed by DNA sequencing. All DNA sequencing was
performed on an ABI sequencer by the DNA core facility at the Burnham
Institute (La Jolla, CA).
 |
RESULTS |
Examination of the Expression of CD45 cDNA
Constructs--
Following reports of expression of CD45 from cDNA
plasmids, pARV/CD45 in thymic lymphoma cells (38), and pML84 and pML171 in transgenic mice (40), we tested expression of CD45 from these transgenes in the T cell hybridoma line, 27J. The full-length CD45
transgene of pARV/CD45 is under the control of the constitutive, retroviral 5' long terminal repeat promoter, but the only discernible poly(A) site in the construct lies in the 3' long terminal repeat ~2.4 kb downstream and beyond the neomycin gene. In contrast, the
transgenes of pML84 and pML171, CD45RABC and
CD45RO, respectively, are regulated by the thymus-specific,
proximal lck promoter, and the human growth hormone gene
provides the poly(A) signal. To facilitate our analysis of
transgene-produced mRNA and protein, we replaced the CD45.2
allogenic determinants of all three cDNAs with sequences specifying
the CD45.1 allele.
Stably transfected populations were generated using these constructs
and were analyzed for CD45.1 expression by flow cytometry. No CD45.1
protein expression from the three cDNA constructs was detected in
any of the transduced cell populations (Fig.
2). Since the CD45 cDNA in plasmids
pML84 and pML171 is under the control of a thymus-specific promoter,
the lack of expression from these plasmids may be due to the inability
of the promoter to function in the cytotoxic T cell hybridoma line.
Therefore, using pML84 and pML171, we produced stably transfected cell
populations in the thymic lymphoma line, BW5147, and employed flow
cytometry to analyze the populations for CD45.1 expression. No CD45.1
protein was detected in these transfected cells (Fig. 2).

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Fig. 2.
Flow cytometry analysis of cDNA
transfectants. The cell line is indicated above each
column. The histograms are labeled according to the plasmid used to
generate the stable cell populations. The cells were stained with
either the FITC-labeled isotype control (isotype), FITC anti-CD45.1
(CD45.1), or FITC anti-CD45.2 (CD45.2) antibodies. The individual peaks
are labeled with the antibody used for staining.
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The site of integration, levels of polyadenylation, transport to the
cytoplasm, and stability of the mRNA are among the factors that can
affect the expression from transgenes, and the structure of the
transgene itself influences these elements (49-53). To better understand possible reasons behind the difficulty obtaining CD45 protein expression from cDNA constructs, we investigated the roles the 5', 3', and intron regions play in regulation.
Analysis of the 3'-Untranslated Region of CD45--
To identify
regulatory elements in the 3'-UTR of CD45, the 3'-UTR was fused
downstream of the
-galactosidase coding region, and the effect on
gene expression was analyzed following transient transfection into
tissue culture cells. The plasmids constructed for this study are shown
in Fig. 1A. The SV40 small t/poly(A) site normally
regulating
-galactosidase mRNA processing in pCMVSPORT
gal (Fig. 1A) was replaced by the entire 3'-UTR of CD45 plus 750 bp of sequence downstream from the end of the mRNA (3) (pSPORT-XR; Fig. 1A). A smaller region beginning 887 bp downstream from
the termination codon and extending 750 bp beyond the polyadenylation site was subcloned in front of the small t/poly(A) site (pSPORT-EA, Fig. 1A) to determine whether the 3' region had any effect,
positive or negative, on the function of an existing poly(A) sequence. pSPORT-BGA (Fig. 1A), in which the SV40 small t/poly(A) site
was replaced with the bovine growth hormone poly(A), and pSPORT-(
A) (Fig. 1A), in which all poly(A) sites were removed, served
as controls for these experiments.
Initially, the plasmids were transfected transiently into various
lymphocyte cell lines (S49, 27J, and Jurkat). However, no
-galactosidase activity above background was detected (data not shown). This result is most likely due to the low transfection efficiency of these cells (S49 and Jurkat) or to high levels of endogenous
-galactosidase activity (27J). 293 cells, on the other hand, exhibit a high frequency of transfection and have low levels of
endogenous
-galactosidase activity. Therefore, the analysis of the
CD45 3' region on transgene expression was completed using the 293 cell line.
-Galactosidase activity was assayed from cellular extracts isolated
48 h after transfection. The results from four separate transfections were averaged and are summarized in Table
I. The
-galactosidase levels observed
in cells transfected with pSPORT-XR, which contains the 3'-UTR and
sequences downstream of the poly(A) site, were essentially equivalent
to the activity seen in those harboring the parental vector,
pCMVSPORT
gal. Introduction of the EcoRI fragment
containing CD45 3'-UTR sequences upstream of the SV40 small t/poly(A)
did not alter
-galactosidase expression substantially (pSPORT-EA;
Table I). In fact, a slight increase in
-galactosidase levels was
reproducibly observed in cells carrying this construct. Removal of all
poly(A) sequences (pSPORT-(
A)) resulted in a significant reduction in
-galactosidase activity, and the bovine growth hormone poly(A)
(pSPORT-BGA) restored activity.
Analysis of the Effect of Different 5' Sequences on CD45 cDNA
Expression--
To investigate how 5' sequences might influence
expression, the cDNA encoding the full-length CD45 isoform (the
456789 isoform) was placed under the control of either the
tissue-nonspecific CMV promoter (pCMVT200; Fig. 1B) or the
leukocyte-specific human LFA-1 (CD11a) promoter (pLFAT200; Fig.
1B). Both transgenes utilize the CD45 poly(A) signal
sequence plus 750 bp of sequence downstream from the mRNA
termination site. In order to follow easily the expression of the CD45
from the transgene in transfected cells, the CD45 cDNA constructs
were prepared containing the CD45.1 allele sequence. The CD45 cDNA
in these constructs was sequenced to confirm an intact coding region.
Transfections were performed using the T-cell hybridoma line, 27J, and
the B lymphocyte cell line, 70Z/3. Both of these cell lines naturally
express the CD45.2 but not the CD45.1 allele (Fig.
3). Stable transfectants were generated, and protein expression from the transgenes was monitored using flow
cytometry and a CD45.1 allele-specific monoclonal antibody. CD45.1
protein production was not detected from either construct in the
transfected cell populations (Fig. 3).

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Fig. 3.
Flow cytometry analysis of cell lines
transfected with pLFAT200 and pCMVT200. The cell line tested is
shown above each column. The transfection treatment is
indicated above each histogram. The antibodies used for
staining are given below the histograms (CD45.1 FITC, CD45.2
FITC).
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To determine whether there was a corresponding lack of transcription
from the transgenes, total RNA was isolated from the transfected cells
and analyzed with RT-PCR using allele-specific primers. The transfected
27J and 70Z/3 cell populations transcribed CD45 mRNA from both the
pCMVT200 and pLFAT200 transgenes (data not shown). Therefore, the CD45
cDNA constructs, pCMVT200 and pLFAT200, produce some mRNA in
both T and B cells, but detectable protein levels are not observed.
Since the sequence of the 5'-end of the transgenes extending from the
ClaI site across the XbaI junction (exon 1b to
exon 9) was identical to published sequence, the lack of protein is not
likely to be the result of a mutation introduced during construction.
RT-PCR is a more sensitive assay for expression compared with flow
cytometry. Therefore, the transgenes appear to be producing small
amounts of mRNA that can be detected with RT-PCR, but the protein
expressed, if any, is below the sensitivity of flow cytometry.
Analysis of CD45 Minigene Expression--
Reports of transgene
expression have indicated that the presence of intron sequences can
increase gene expression (49-51). Since one of the interesting
properties of the endogenous CD45 gene is the production of multiple
isoforms through mRNA splicing, a CD45 expression construct that
included the alternatively spliced exons and associated introns might
not only exhibit increased levels of expression but also would provide
the possibility of producing the various isoforms. Therefore, a CD45
minigene, encoding the CD45.1 allele, was generated and placed under
the control of the LFA-1 promoter (pLFAiT200; Fig. 1C). In
this plasmid, genomic DNA carrying the alternatively spliced exons 4-8
is flanked by two cDNA regions that span exons 1b-3 and 9-33.
The cell lines 27J and 70Z/3 were transfected with
pLFAiT200, and stable populations expressing the neomycin resistance
gene were selected. Flow cytometry with allele-specific antibodies was
used to detect CD45 protein expression from the transgene. CD45.1
expression was dramatically increased in the transfected populations
compared with the nontransfected control, suggesting that the pLFAiT200
minigene was producing substantial levels of CD45 protein (Fig.
4).

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Fig. 4.
Flow cytometry analysis of pLFAiT200
transfectants. The cell line is indicated above each
column. The individual peaks of the bottom histogram are
labeled with the transfection treatment. The staining antibodies are
shown below the histograms (CD45.1 FITC, CD45.2 FITC).
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RT-PCR analysis of RNA isolated from the B and T cell transfectants was
used to evaluate CD45 isoform expression from the minigene. Two
CD45.1-specific mRNA species, corresponding to the 789 and 89 isoforms, were detected in the 27J transfected cells (Fig.
5A, lane
4). These are the same splice variants as produced by the
CD45.2 endogenous allele (Fig. 5A, lane
3). The pattern of CD45 exon usage from the transgene and
the endogenous gene also was identical in 70Z/3-transfected populations
(Fig. 5B, lanes 3-5). The predominant
isoform was 456789, with minor amounts of the 56789 form detected.
No amplification products were seen using the CD45.1-specific primer
with RNA isolated from nontransfected cells (Fig. 5, A and
B, lane 2). Although the ratio of the
two isoforms produced from the transgene and from the endogenous gene differs slightly in the 27J transfectants, both the 27J and 70Z/3 transfected populations express the same isoforms from the transgene as
are expressed from the endogenous gene. Thus, the minigene carries the
regulatory elements necessary for cell type-specific splicing.

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Fig. 5.
RT-PCR products from RNA isolated from T and
B cell populations transfected with pLFAiT200. In both
A and B, lane 1 contains
molecular weight markers. Amplification products in all
even-numbered lanes of both panels were generated
using the CD45.1 allele-specific antisense primer, and the
odd-numbered lanes contain fragments amplified with the
CD45.2-specific primer. In A, the RNA was isolated from 27J
cells either nontransfected (lanes 2 and
3) or transfected with pLFAiT200 (lanes
4 and 5). B, RNA was from 70Z/3 cells
either nontransfected (lanes 2 and 3)
or transfected with pLFAiT200 (lanes 4 and
5). Lanes 6 and 7 are no
RNA, negative controls.
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Control of the Minigene with CD45 5' Upstream Sequences--
To
identify upstream sequences responsible for regulating CD45 gene
expression and to create a minigene that more closely resembles the
endogenous gene, a
genomic clone-containing sequence extending 19 kb upstream from the start of CD45 translation was utilized. The 19-kb
region was inserted in place of the LFA-1 promoter in pLFAiT200,
creating pCD45FiT200 (Fig. 1D). In the same fashion,
minigene constructs containing 5' truncations of the 19-kb CD45
upstream region were generated. pCD45P2iT200, pCD45PiT200,
and pCD45EiT200 extend 3.5 kb, 2.2 kb, and 839 bp, respectively,
upstream from the translational start site (Fig. 1D).
Stable transfectants of the cell line 27J were generated with each of
these transgenes. For a positive control, the cells were transfected
with pLFAiT200. Expression from the transgenes was monitored by flow
cytometry with the allele-specific antibodies (Fig.
6). As observed from previous
experiments, CD45.1 protein was readily detected in the cells
transfected with pLFAiT200. Compared with the isotype control, a
minimal shift in the mean fluorescence intensity of CD45.1 was detected
in cells harboring the pCD45EiT200 and pCD45FiT200 transgenes, while in
pCD45PiT200 transfectants the CD45.1 peak exhibited a slightly more
significant but still minor change. No CD45.1 protein expression was
observed in cells containing pCD45P2iT200.

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Fig. 6.
Flow cytometry of the T cell populations
transfected with the CD45 upstream constructs. The plasmids used
to generate the stable cell populations are given at the top
of each histogram. The cells were stained with either the isotype
control FITC anti-THP (isotype), FITC anti-CD45.1 (CD45.1), or FITC
anti-CD45.2 (CD45.2) antibodies. The individual peaks are labeled with
the antibody used for staining.
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Allele-specific RT-PCR experiments were carried out using these 27J
transfected cell populations. The expected 789 and 89 isoform
transcripts were expressed from all four constructs, and no fragments
corresponding to CD45.1 transcripts were detected in nontransfected
cells (data not shown)
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DISCUSSION |
For the analysis of the molecular basis of the developmental and
functional properties of CD45, a reliable expression system is needed.
However, obtaining stable high level expression from CD45 cDNA
constructs has been difficult and inconsistent (41). Therefore, in an
effort to optimize expression from CD45 cDNAs, we tested 5', 3',
and intron regions for their effect on transgene expression.
A number of examples are known for translational control of mRNA
through sequences in the 3'-UTR (43, 44, 54). In many cases, these
regulatory elements interact with positive or negative trans-acting
factors, thereby controlling mRNA stability or localization (43,
44, 54). To assess the possible role of the CD45 3'-UTR in expression,
the 3'-UTR and downstream sequences were fused 3' of the
-galactosidase coding region. Our results show that the CD45
polyadenylation signal was fully functional in conjuction with the
-galactosidase reporter gene. Furthermore, the presence of the CD45
3' sequences had no effect on the levels of
-galactosidase expressed
in the transient transfection assays, suggesting that no negative
cis-acting regulatory factors are present. Since these experiments
measured the effect on a heterologous gene (
-galactosidase) in a
nonlymphocyte system, the possibility of leukocyte-specific controls
was not eliminated by this analysis.
Stable lymphoid cell populations generated by transfection with
constructs containing CD45.1 coding sequences show dramatic differences in levels of CD45.1 protein expressed. pLFAT200 and pLFAiT200 only differ in the inclusion of the six introns present in
pLFAiT200. The addition of the intron sequences led to a substantial increase in protein expression as measured by flow cytometry. Both the
LFA-1 promoter and the strong CMV promoter did not produce detectable
protein levels in the CD45 cDNA constructs without the introns.
Although the exact mechanism by which the introns affect expression is
not known, it has been shown that introns can contain enhancers
(55-59), transcriptional silencers (60, 61), and elements controlling
cell-specific expression (61-64). Furthermore, splicing can influence
the levels of cytoplasmic mRNA through posttranscriptional mRNA
processing (49). Examples of this processing include increased nuclear
mRNA stability, increased polyadenylation, and increased transport
to the cytoplasm. Utilizing RT-PCR, we found that the cell populations
transfected with the CD45 cDNA constructs all produced RNA from the
transgene, supporting the possibility that a posttranscriptional
mechanism is responsible for the intron-related increase in expression.
RT-PCR also showed that T and B cells transfected with pLFAiT200
produced the distinctive isoforms characteristic of each cell type.
Therefore, the controls for correct splicing of the alternative exons
lie within the pLFAiT200 construct. Previous splicing studies also
concluded that the sequences within the introns and exons in this
region are sufficient to regulate alternative splicing (65-68).
Finally, it should be noted that both pLFAT200 and pLFAiT200 utilize an
identical CD45 3' region extending from exon 33 to 750 bp downstream
from the polyadenylation site, suggesting that this sequence contains
no cis-acting elements responsible for controlling expression in
lymphoid cells.
Although the CD45.1 protein levels were enhanced in
pLFAiT200-transfected cells, they were below endogenous CD45 expression in both cell types. One simple explanation would be that the transgene is subject to integration-dependent positional effects.
However, since these transfected populations are not clonal, it would
be expected that a positional effect in a few cells would be averaged out over the entire population. Also, the lower level of expression from the transgene may be due to particular properties of the cell
lines used in this study. Another potential explanation is related to
the importance of the introns for expression. The included introns are
located between exons 3 and 9 in pLFAiT200, and the remainder of the
construct (exons 9-33) is strictly cDNA. Therefore, additional
splicing events that could contribute to endogenous CD45 gene
expression cannot occur.
Perhaps the most important feature of the construct affecting
expression levels is the human LFA-1 promoter that controls the
transgene expression in mouse cell lines. The expression level may
reflect a diminished ability of the human promoter to interact with a
murine regulatory factor. Furthermore, the protein levels of endogenous
CD45 are very high, ~10% of lymphocyte surface proteins (69), and
the human LFA-1 promoter may not be able to drive expression to the
same levels. The LFA-1 promoter, however, contributes several features
that make it an attractive choice for a heterologous promoter in these
studies. First, the CD11a gene is expressed in a cell type-specific
pattern similar to CD45 (46, 70, 71). Second, in mice the human LFA-1
promoter directed transgene expression in a pattern parallel to
endogenous murine CD11a (71). However, T cell developmental signals do
play a role in the regulation of CD11a. Low level expression of CD11a
is thought to characterize naive T cells, while expression of high
levels of CD11a (CD11abright) is a property of memory T
cells (72). The 27J T cell hybridoma line exhibits the
CD11abright phenotype (data not shown).
In an effort to improve expression, the natural CD45 upstream region
was investigated. Examination of the sequences upstream from the CD45
translation initiation sites reveals neither a consensus TATA box near
positions
30 to
25 nor a pyrimidine-rich initiator usually located
near the transcription start site. Typically, recognition sites for
DNA-binding transcription factors lie upstream (
50 to
200 bp
relative to the transcription start) from these core promoter elements
(73). In addition, transcriptional enhancers and silencers located far
upstream or downstream of a gene's promoter may regulate transcription
(73, 74). Although the CD45 upstream region does contain canonical
binding sites for many transcription factors, the possible role of
these sites in CD45 regulation has not been reported. To test the
promoter activity of CD45 upstream sequences, the CD45 minigene was
placed under the control of different CD45 upstream regions, extending
to 19,000 bp 5' of the translational start. Stably transfected cell
populations were generated with these transgenes and mRNA, but
little or no protein, was detected. The CD45 protein levels did not
vary significantly between these various CD45 upstream constructs,
indicating that the low level of expression most likely is due to the
absence of an enhancer. One previously identified control element, a TC
box located between exons 1b and 2 that acts as a tissue-specific
initiator for a minor transcriptional start position 3' of exon 2 (15),
is missing in our constructs. However, the two major transcriptional
start sites at exons 1a and 1b with surrounding sequence are present in
these constructs. An intriguing candidate for the location of
additional transcriptional control elements is the 50-kb intron between
exons 2 and 3, an intron whose unusual size and location is conserved
between species.
Our experiments indicate that regulation of CD45 is a complex
process requiring elements that lie within the introns of the gene and
possibly well outside of the coding region. The 3'-UTR and associated
downstream sequences contain no positive or negative regulatory
elements. The CD45 region up to 19 kb upstream of the transcriptional
start is not sufficient to produce readily detectable levels of
expression. However, the intron sequences, located within the genomic
DNA segment encoding the alternatively spliced exons, enhance
expression of the transgene dramatically and contain the controls
necessary for correct splicing of the alternative exons. Using the
pLFAiT200 minigene, we were able to reproducibly generate stable
lymphoid cell populations producing correctly spliced CD45.1 mRNA
and protein.