|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 663-672
By
From the Institute of Immunobiology, University of Freiburg,
Freiburg, Germany; and Novartis, Basel, Switzerland.
The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a
member of the human carcinoembryonic antigen (CEA) family encoded by
the CGM6 gene that is exclusively expressed in neutrophils and
eosinophils. No murine counterpart is known to exist. We have analyzed
a cosmid containing the complete CGM6 gene. The coding sequence
is contained within six exons spanning a 16.5 kb region. The main
transcriptional start site was mapped to a tight cluster between
nucleotides -95 and -101 relative to the translational start site. As
with other members of the CEA gene family, no typical TATA or CAAT-box
sequences were found in the CGM6 gene. Transgenic mice were
established with the cosmid insert. CD66b expression is first seen in
the fetal liver on day 12.5 of mouse embryonic development, and it
first appears in the bone marrow at day 17.5. Northern blot analysis
showed that CD66b transcripts are confined to the bone marrow of adult
mice, whereas immunohistochemistry also showed CD66b-positive
granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone
marrow and spleen cells showed CD66b expression to be exclusive to
granulocytes. Thus, all the elements necessary for regulating
granulocyte-specific expression are present within this cosmid clone.
These mice could provide a model for transplantation and for
inflammation studies using CD66b as a granulocyte-specific marker.
CD66b (FORMERLY CD67) HAS been
characterized as a granulocyte-specific activation
antigen.1 Using specific antibodies, CD66b has been located
on the surface of neutrophilic and eosinophilic granulocytes at late
stages of differentiation. To our knowledge, it is not known whether or
not CD66b is expressed by basophils. In neutrophils, it is mainly found
in the secondary granules within the cytoplasm, with lower amounts on
the plasma membrane.2 On activation of granulocytes with
various agents, the amount of CD66b on the plasma membrane is rapidly
upregulated, presumably through release from the intracellular
granules.3,4 By measuring stimulation of oxidative burst,
granulocyte activation could be achieved through cross-linking CD66b
surface molecules using F(ab CD66b is synonymous with the nonspecific cross-reacting antigen-95
(NCA-95), a member of the carcinoembryonic antigen (CEA) family.8 The CEA gene family consists of 29 genes, of which 18 are transcriptionally active. The genes are divided into three main
subgroups: the CEA subgroup, the pregnancy-specific glycoprotein (PSG)
subgroup, and a subgroup of pseudogenes.9 Through molecular cloning, NCA-95 was found to be the product of the CGM6 gene, a
member of the CEA subgroup.10,11 The derived protein
structure of NCA-95 shows an immunoglobulin variable-like N-terminal
domain, followed by two immunoglobulin constant-like domains. It is
attached to the membrane through a glycosylphosphatidyl inositol (GPI) anchor. Some other members of the CEA family, ie, CGM1 (CD66d), biliary
glycoprotein (BGP; CD66a), and NCA-50/90 (CD66c) are coexpressed with
NCA-95 on granulocytes. Whereas CGM1 shows an identical expression pattern to NCA-95, BGP and NCA-50/90 are also present in other normal
tissues, eg, in epithelial cells lining the gastrointestinal tract.12,13 Although its in vivo function is unknown, CD66b shows heterophilic cell adhesion properties with the closely-related CD66c molecule, which is coexpressed in granulocytes.14
Transcriptional regulation specific for the myeloid lineage has been
well investigated,15-17 but less is known about specific transcriptional regulation in the granulocytic lineage, ie, after separation from the monocytic differentiation pathway. Due to its
expression pattern, investigations on the regulation of expression of
the CGM6 gene should help in a better understanding of gene regulation in later development of neutrophils and in eosinophils. For
this reason, we are interested in locating the regions responsible for
regulating the granulocyte-specific expression pattern of the
CGM6 gene. As a basis for such investigations, we have
characterized a cosmid clone containing the putative promoter region,
as well as the complete coding sequence of the CGM6 gene,
through restriction endonuclease and exon mapping. After determining
the transcriptional start site, we have compared putative promoter
sequences with corresponding regions from other members of the CEA gene
family. Finally, we have established transgenic mice to determine
whether all the cis-regulatory elements necessary for its
tissue-specific expression pattern are present in this cosmid clone.
Isolation and Characterization of a Cosmid Clone Containing the CGM6
Gene.
Determination of the Transcriptional Start Site and Promoter
Analyses of CGM6
Generation of CGM6 Transgenic Mice Cosmid F19632 DNA was digested with SfiI and the 42-kb insert containing the complete CGM6 insert including 0.3 kb 5 -
and 0.1 kb 3-vector sequences was isolated from a 0.7% agarose
gel by the freeze-squeeze method and used for microinjection into fertilized mouse oocytes derived from C57BL/6 × CB6 F1 mice (Ciba Animal Breeding Center, Basel, Switzerland) as described
elsewhere.21,22 A mouse line was established from founder
animals by backcross mating with C57BL/6 mice (Zentralinstitut
für Versuchstierzucht, Hannover, Germany). Transgenic mice are
routinely identified by PCR, using the CGM6-specific primers
and PCR conditions essentially as described elsewhere, but with an
annealing temperature of 58°C.23
Southern and Northern Blot Analyses DNA was isolated from the thymuses of CGM6 transgenic and C57BL/6 mice. A total of 10 µg of each were digested with EcoRI, size-separated by gel electrophoresis, and blotted onto a positively charged nylon membrane. To estimate the number of the CGM6 transgene copies, C57BL/6 DNA was spiked with CGM6 cosmid DNA, corresponding to 25 to 150 copies per haploid genome. After hybridization with [32P]-labeled EcoRI-digested F19632 cosmid DNA, the blot was washed twice in 0.1× SSPE (1× SSPE is 0.18 mol/L NaCl, 10 mmol/L sodium phosphate, pH 7.4, 1 mmol/L EDTA), 0.1% sodium dodecyl sulfate (SDS) for 30 minutes at 68°C. Autoradiography was performed for 48 hours.
Immunohistochemical Analyses To determine tissue expression of CD66b by immunohistochemical methods, organs were isolated from adult transgenic mice, which had been anesthetized with Forene (Abbott GmbH, Wiesbaden, Germany) and killed by cervical dislocation. The organs were embedded and cryosectioned as described elsewhere.24 Rabbit anti-NCA antiserum was kindly provided by Dr Fritz Grunert (Institute of Immunobiology, Freiburg, Germany) and used at a 1:200 dilution in phosphate-buffered saline (PBS). This antiserum was developed against NCA-50/90 extracted from a breast tumor and was found to cross-react with other human CEA family members, including CD66b, but not with mouse CEA family members (F. Grunert, personal communication, May 1997). Binding of the primary antibody was followed by incubation with horseradish peroxidase-conjugated, goat antirabbit IgG at a dilution of 1:100 (Sigma-Aldrich, Deisenhofen, Germany). The staining reaction was performed using the substrate 3,3-diaminobenzidine tetrahydrochloride. Sections were
counterstained with hematoxylin. Staged nonfixed embryos were treated
likewise. Earlier stage embryos (embryonic day 8.5) were sectioned in
toto. Staged embryos were obtained from mating nontransgenic females
overnight with transgenic males. Midday after mating was designated
embryonic day 0.5.
FACScan Investigations For identification of the cell population that express CD66b, three-color fluorescence FACScan analyses was performed on cells isolated from the bone marrow of mice transgenic for CGM6 as described.27 Erythrocytes were lysed by resuspending the isolated cell populations in ice-cold Gey's solution (130 mmol/L NH4Cl, 5 mmol/L KCl, 0.8 mmol/L Na2HPO4, 0.18 mmol/L KH2PO4, 5 mmol/L glucose, 10 mg/L phenol red, 0.1 mmol/L MgCl2, 0.03 mmol/L MgSO4, 0.1 mmol/L CaCl2, 13.5 mmol/L NaHCO3), which was immediately underlayered with fetal bovine serum and the intact cells were spun down and resuspended in fluorescence-activated cell sorting (FACS) buffer. The number of intact cells was determined in a hemacytometer after adding Trypan blue and equal cell numbers (2 to 5 × 105) were incubated with the labeled antibodies in concentrations ranging from 0.17 to 1.0 µg/mL as determined in titration experiments. The fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (MoAb), antihuman CD66b (clone 80H3: Coulter-Immunotech, Hamburg, Germany) was used to identify the CGM6 gene product. The following phycoerythrin (PE)-conjugated MoAb was used to identify the main leukocyte populations: for B lymphocytes, antimouse B220, clone RA3-6B2; for T lymphocytes, antimouse Thy-1.2, clone 30-H12; for myeloid cells, antimouse GR-1, clone RBG-8CS. All were obtained from PharMingen (Hamburg, Germany). Single and double labelings were performed in FACS buffer (PBS containing 3% fetal bovine serum), whereby for the second incubation, FACS buffer without antibody was used for the single labelings to ensure identical conditions. Before FACScan analysis, 5 µg/mL propidium iodide (Fluka, Deisenhofen, Germany) was added to allow subtraction of dead cells.
Cosmid Clone F19632 Contains the Complete CGM6 Gene Digestion of cosmid clone F19632 with SfiI resulted in a 5-kb vector fragment and a 42-kb fragment encompassing human genomic DNA flanked by 0.3 kb vector at the 5 end and 0.1 kb vector 3 to the CGM6 gene. A complete restriction endonuclease map,
including the exon positions within this SfiI fragment is shown
in Fig 1A. The CGM6 gene consists
of six exons that span 16.5 kb. There is a strong correlation between
the exon and derived protein domain borders for this
gene.10 CGM6 is flanked by 13 kb of 5- and approximately 12.5 kb of 3-genomic sequences.
The CGM6 Gene Promoter Is Most Similar to PSG Gene Promoters The transcriptional start site, as determined by primer extension experiments shows a tight cluster at nucleotide positions -95 to -101, with respect to the translational start (Fig 1B and C). An additional start site, not found in the control RNA samples was located at nucleotide -69, although this could also represent a strong stop site during the primer extension reaction, due to secondary structures in the template. Although the sequence reaction in Fig 1C is rather weak, additional experiments proved the exact positions of the transcriptional start sites. DNA sequencing of a 1,740-bp SstI fragment that contains the first exon and 1210 nucleotides upstream from the translational start site is shown in Fig 1B. Along with the transcriptional start cluster, a potential SP-1 binding element is marked at nucleotide positions -147 to -140 relative to the translational start site.28 A second element is indicated at nucleotide positions -255 to -247 relative to the translational start site, which shows a strong similarity to a C-EBP binding site.29 A repetitive simple sequence is found at nucleotides -356 to -309. As for other members of the CEA gene family, CGM6 lacks recognizable TATA or CAAT-box sequences at the expected positions. Sequence comparisons by dot matrix analyses have been performed between the promoter and 5-upstream regions of
various members of the CEA gene family. Surprisingly, the longest region of homology is found to the corresponding region in genes from
the PSG subgroup (Fig 2A), which are mainly
expressed in the syncitiotrophoblast of the placenta.13
This region covers 800 nucleotides upstream from the translational
start site. The repetitive simple sequence region found in the
CGM6 gene promoter is also present in PSG gene
promoters, which is visible in this alignment analysis (Fig 2A). In
comparison with gene promoters from other members of the CEA subgroup,
ie, CGM1 (Fig 2B), which like CGM6 is exclusively
expressed in granulocytes, NCA (Fig 2C), which is expressed in
granulocytes and epithelial cells, CEA (Fig 2D), whose
expression is exclusive to certain epithelial cells and BGP
(data not shown), that is expressed in granulocytes, epithelial cells
and other tissues, this region of homology is limited in all cases to
approximately 240 nucleotides upstream from the translational start
site. Two of the CEA family members that are coexpressed in
granulocytes (NCA and CGM1) are homologous to each
other over the whole 800 nucleotide promoter region (Fig 2E), whereas
the CEA and NCA gene promoters are less well conserved
(Fig 2F).
Establishment of Mice Transgenic for the Human CGM6 Gene To determine whether the CGM6 cosmid clone contains all the regulatory elements that are necessary for granulocyte-specific expression, the 42-kb genomic fragment was used to establish transgenic mice. From 16 founder mice that were born, only one was transgenic for the CGM6 gene, as determined by Southern blot analysis of EcoRI-digested mouse tail DNA, hybridized with a radioactively-labeled EcoRI digest of the complete cosmid clone F19632. The CGM6 cosmid probe shows no cross-hybridization with C57BL/6 genomic mouse DNA (Fig 3, lane 1; see also Fig 1A). In the CGM6-positive strain, the transgene was found to contain all of the EcoRI fragments that were present in the original cosmid, apart from the 1.2-kb and the 3.75-kb vector fragments and the 3.2-kb and 4.3-kb end fragments (Fig 3, lane 2). However, a new 7.5-kb EcoRI fragment, resulting from the fusion of the 3.2-kb 5-end EcoRI/SfiI fragment with the 4.3-kb, 3-end EcoRI/SfiI fragment of
the cosmid insert, is seen in the transgene, which comigrates with an
internal EcoRI fragment (Fig 3, lane 2). This fusion fragment
indicates the existence of multiple cosmid inserts in a tandem
head-to-tail orientation. After inclusion of different cosmid copy
numbers with C57BL/6 genomic DNA, we determined a total of 50 to 75 intact transgene copies to have become integrated in this mouse strain
(Fig 3, lane 3 and titration data not shown).
CD66b Is Expressed Exclusively in Granulocytes of CGM6-Transgenic Mice Northern blot analyses.
To investigate the presence of transcripts from the CGM6 gene,
total RNA from 14 different organs of a CGM6 transgenic mouse was analyzed for the presence of CGM6 mRNA. A positive signal was only
seen in RNA from bone marrow (Fig 4, lane
3). All other organs tested were negative for CGM6 mRNA. Although
slightly degraded, the hybridizing mRNA in bone marrow shows a size
that is similar to that reported elsewhere for human CGM6
transcripts.10 This mRNA was not present in the bone marrow
of a nontransgenic sibling (data not shown). A positive signal was seen
in all lanes after hybridization with a probe that detects mouse
Immunohistochemical analyses. Immunohistochemical staining was performed on peripheral blood smears and 12 different organs (brain, tongue, stomach, small intestine, large intestine, spleen, thymus, lung, uterus, ovary, kidney, and liver) of adult CGM6-transgenic mice using a polyclonal rabbit anti-NCA serum that cross-reacts with CD66b. This antiserum was used because the CD66b-specific mouse MoAb 80H3, showed a high background staining on the mouse tissues (data not shown). It is obvious from Fig 5D that the cells staining positive in peripheral blood smears are polymorphonuclear and strongly resemble granulocytes, whereas the lymphocytes are negative. Positive staining was seen in the red pulp areas of the spleen (Fig 5A). No staining was seen in any tissues of nontransgenic sibling mice, shown in Fig 5B for the spleen, confirming that the anti-NCA serum used does not cross-react with any members of the mouse CEA family. The thymus (data not shown) and lungs (Fig 5C) of transgenic animals also showed labeled cells. All other organs, including the adult liver, were negative. The labeled cells in CGM6-transgenic mice often strongly resembled granulocytes in their morphology, although this could not be unequivocally determined in tissue sections.
FACScan investigations. In an attempt to characterize the CD66b-expressing cells in CGM6-transgenic mice, FACScan analyses were performed on cells isolated from the bone marrow from mice in the fourth filial generation after backcrossing with C57BL/6 mice. The results of one experiment are shown in Fig 6. The forward/sideward scatter plot of the total cell population shows the three main leukocyte populations (Fig 6A), which were analyzed as a group for CGM6 expression. The remaining signals that represent debris and possible cell aggregates were excluded from the analyses. In the case of the T lymphocyte analysis (Fig 6H), only leukocytes within the lymphocyte gate (Fig 6A) were tested for antibody recognition so as to increase the number of T cells analyzed. Double-labeling experiments were performed using a FITC-labelled, CD66b-specific MoAb and PE-labelled rat antibodies specific for the mouse myeloid marker, GR-1 (Fig 6E and F), the B-lymphocyte marker, B-220 (Fig 6G) and the T-lymphocyte marker, Thy-1 (Fig 6H), respectively. As bone marrow cells from nontransgenic mice showed no signal with the CD66b-specific antibody (Fig 6F), cross-reactivity with murine CEA family members on mouse leukocytes, as well as possible binding by Fc-receptors, could be ruled out. These results showed that CD66b is not expressed on T, or B lymphocytes in the transgenic mice, but a large proportion of the GR-1-expressing myeloid cells also express CD66b (Fig 6E, upper right quadrant). Forward/sideward scatter analysis of these double-positive cells confirmed their identity as granulocytes (Fig 6C). Interestingly, some cells exist, which only express GR-1, but not CD66b (Fig 6E, upper left quadrant). Based on their forward/sideward scatter properties, these cells consist mainly of monocytes (Fig 6B). Cells also exist, which only express CD66b, but not GR-1 (Fig 6E, lower right quadrant), although these do not represent a separate population to the doubly-labeled cells. Analysis of this CD66b-positive, GR-1-negative group of cells using forward/sideward scatter confirmed that these cells are also granulocytes (Fig 6D), although there is a slight shift in the sideward scatter towards the monocyte population. These analyses suggest that the granulocytes in the transgenic animal are heterogenous for GR-1 expression.
As a first step towards investigating mechanisms controlling granulocyte-specific expression, we have analyzed a cosmid clone containing the complete CGM6 gene, which encodes the human granulocyte marker, CD66b/NCA-95.10 Furthermore, we have developed CGM6-transgenic mice with this cosmid that express CD66b exclusively in granulocytes. Experimental transgenic model systems using genomic fragments from YAC clones are currently being developed to analyze tissue-specific, cis-regulatory sequences in various genes.30 As seen from these investigations, similar studies are possible using cosmid inserts. We previously reported on mice that are transgenic for a cosmid insert containing the complete human CEA gene, which is exclusively expressed in epithelial cells. Those mice show a conserved spatiotemporal CEA expression pattern when compared with humans, despite the fact that mice do not have an endogenous CEA gene.24,31 These findings suggested the feasibility of determining the presence of tissue-specific cis-regulatory elements in the closely-related CGM6 gene. Although only one CGM6-transgenic line was developed, which contains 50 to 75 copies of the intact cosmid insert in a head-to-tail orientation, the granulocyte-specific expression pattern was found to be identical to that observed in humans.13 This indicates that all of the regulatory elements, which convey tissue-specificity, are present within this cosmid insert and that no additional effects due to the site of genomic integration can be registered. As mice apparently do not have counterparts to any of the GPI-linked members of the CEA family, including CD66b and CEA, it is rather surprising that the expression pattern is identical.32 However, the cis-regulatory elements present within these genes are obviously recognized and interact correctly with the murine trans-acting factors. Thus, the in vivo investigations on the CGM6 transgenic mice provide a basis for the identification of granulocyte-specific cis-regulatory elements and the interacting transcription factors.
Submitted May 13, 1997;
accepted September 15, 1997.
We wish to thank Sabine von Kleist for her continuous support, as well as Rita Carsetti and Craig Stocks for their assistance and guidance with the FACScan analyses. We acknowledge the reliable technical assistance of Margarethe Ditter, Beate Fischer, Dirk Schillinger, and Joachim Grammel.
1. Skubitz K, Micklem K, van der Schoot E: CD66 and CD67 cluster workshop report, in Schlossmann SF, Boumsell L, Gilks W, Harlan J, Kishimoto T, Morimoto C, Ritz J, Springer TA, Tedder TF, Todd RF (eds): Leukocyte Typing V (vol 1). Oxford, UK, Oxford, 1995, p 889 2. Ducker TP, Skubitz KM: Subcellular localization of CD66, CD67 and NCA in human neutrophils. J Leukoc Biol 52:11, 1992[Abstract] 3. Tetteroo PAT, Bos MJE, Visser FJ, von dem Borne AEGKr: Neutrophil activation detected by monoclonal antibodies. J Immunol 136:3427, 1986 4. Kuroki M, Matsuo Y, Kinugasa T, Matsuoka Y: Augmented expression and release of nonspecific cross-reacting antigens (NCAs), members of the CEA family, by human neutrophils during cell activation. J Leukoc Biol 52:551, 1992[Abstract] 5. Lund-Johansen F, Olweus J, Symington FW, Arli A, Thompson JS, Vilella R, Skubitz K, Horejsi V: Activation of human monocytes and granulocytes by monoclonal antibodies to glycosylphosphatidylinositol-anchored antigens. Eur J Immunol 23:2782, 1993[Medline] [Order article via Infotrieve] 6. Andres RY, Schubiger PA, Teifenauer L, Seybold K, Locher JT, Mach J-P, Buchegger F: Immunoscintigraphic localization of inflammatory lesions: Concept, radiolabelling and in vitro testing of granulocyte specific antibody. Eur J Nucl Med 13:582, 1988[Medline] [Order article via Infotrieve] 7. Collet B, Maros S, Moisan A, Le Cloirec J, Moinereau M, Aumaitre E, Toujas L, Bourguet P: 111Indium-F(ab)'2-NCA 102 monoclonal antibody: In vitro study of a specific agent for the detection of inflammatory foci. Nucl Med Biol 20:175, 1993[Medline] [Order article via Infotrieve] 8. Thompson JA, Grunert F, Zimmermann W: Carcinoembryonic antigen gene family: Molecular biology and clinical perspectives. J Clin Lab Anal 5:344, 1991[Medline] [Order article via Infotrieve] 9. Teglund S, Olsen A, Khan WN, Frängsmyr L, Hammarström S: The pregnancy-specific glycoprotein (PSG) gene cluster on human chromosome 19: Fine structure of the 11 PSG genes and identification of 6 new genes forming a third subgroup within the carcinoembryonic antigen (CEA) family. Genomics 23:669, 1994[Medline] [Order article via Infotrieve]
10.
Berling B,
Kolbinger F,
Grunert F,
Thompson JA,
Brombacher F,
Buchegger F,
von Kleist S,
Zimmermann W:
Cloning of a carcinoembryonic antigen family member expressed in leukocytes of chronic myeloid leukaemia patients and bone marrow.
Cancer Res
50:6534,
1990 11. Arakawa F, Kuroki M, Misumi Y, Oikawa S, Nakazato H, Matzuoka Y: Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family. Biochem Biophys Res Commun 166:1063, 1990[Medline] [Order article via Infotrieve] 12. Nagel G, Grunert F, Kuijpers TW, Watt SM, Thompson J, Zimmermann W: Genomic organization, splice variants and expression of CGM1, a CD66-related member of the carcinoembryonic antigen gene family. Eur J Biochem 214:27, 1993[Medline] [Order article via Infotrieve] 13. Thompson JA: Molecular cloning and expression of carcinoembryonic antigen gene family members. Tumor Biol 16:10, 1995
14.
Oikawa S,
Inuzuka C,
Kuroki M,
Arakawa F,
Matsuoka Y,
Kosaki G,
Nakazato H:
A specific heterotypic cell adhesion activity between members of carcinoembryonic antigen family, W272 and NCA, is mediated by N-domains.
J Biol Chem
266:7995,
1991
15.
Pang J-HS,
Hung R-Y,
Wu C-J,
Fang Y-Y,
Chau L-Y:
Functional characterization of the promoter region of the platelet-activating factor receptor gene.
J Biol Chem
270:14123,
1995
16.
Clarke S,
Greaves DR,
Chung L-P,
Tree P,
Gordon S:
The human lysozyme promoter directs reporter gene expression to activated myelomonocytic cells in transgenic mice.
Proc Natl Acad Sci USA
93:1434,
1996
17.
Nickel J,
Short ML,
Schmitz A,
Eggert M,
Renkawitz R:
Methylation of the mouse M-lysozyme downstream enhancer inhibits heterotetrameric GABP binding.
Nucleic Acids Res
23:4785,
1995 18. Thompson J, Zimmermann W, Osthus-Bugat P, Schleussner C, Eades A-M, Barnert S, von Kleist S, Willcocks T, Craig I, Tynan K, Olsen A, Mohrenweiser H: Long range chromosomal mapping of the carcinoembryonic antigen (CEA) gene family cluster. Genomics 12:761, 1992[Medline] [Order article via Infotrieve] 19. de Jong PJ, Yokabata K, Chen C, Lohman F, Pederson L, McNinch J, van Dilla M: Human chromosome-specific partial digest libraries in lambda and cosmid vectors. Cytogenet Cell Genet 51:985, 1989 20. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology (vol 1). New York, NY, Wiley, 1992 21. Tautz D, Renz M: An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 132:14, 1983[Medline] [Order article via Infotrieve]
22.
Botteri FM,
van der Putten H,
Wong DF,
Sauvage CA,
Evans RM:
Unexpected thymic hyperplasia in transgenic mice harboring a neuronal promotor fused with simian virus 40 large T antigen.
Mol Cell Biol
7:3178,
1987 23. Thompson J, Mössinger S, Reichardt V, Engels U, Beauchemin N, Kommoss F, von Kleist S, Zimmermann W: A polymerase chain reaction assay for the specific identification of transcripts encoded by individual carcinoembryonic antigen (CEA) gene family members. Int J Cancer 55:311, 1993[Medline] [Order article via Infotrieve]
24.
Eades-Perner A-M,
van der Putten H,
Hirth A,
Thompson J,
Neumaier M,
von Kleist S,
Zimmermann W:
Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern.
Cancer Res
54:4169,
1994 25. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989 26. Alonso SA, Minty A, Bourlet Y, Buckingham M: Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J Mol Evol 23:11, 1986[Medline] [Order article via Infotrieve]
27.
Brombacher F,
Kohler G,
Eibel H:
B cell tolerance in mice transgenic for anti-CD8 immunoglobulin µ chain.
J Exp Med
174:1335,
1991
28.
Kadonaga JT,
Tjian R:
Affinity purification of sequence-specific DNA binding proteins.
Proc Natl Acad Sci USA
83:5889,
1986
29.
Alam T,
An MR,
Mifflin RC,
Hsieh CC,
Ge X,
Papaconstantinou J:
Trans-activation of the alpha 1-acid glycoprotein gene acute phase responsive element by multiple isoforms of C/EBP and glucocoticoid receptor.
J Biol Chem
268:15681,
1993 30. Peterson KR, Clegg CH, Li Q, Stamatoyannopoulus G: Production of transgenic mice with yeast artificial chromosomes. Trends Genet 13:61, 1997[Medline] [Order article via Infotrieve] 31. Keck U, Nédellec P, Beauchemin N, Thompson J, Zimmermann W: The Cea10 gene encodes a secreted member of the murine carcinoembryonic antigen family and is expressed in the placenta, gastrointestinal tract and bone marrow. Eur J Biochem 229:455, 1995[Medline] [Order article via Infotrieve] 32. Zimmermann W: The nature and expression of the rodent CEA families: evolutionary considerations, in Stanners CP (ed): Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Amsterdam, The Netherlands, Harwood (in press) 33. Zhu J, Bennett CA, MacGregor AD, Greaves MF, Goodwin GH, Ford AM: A myeloid-lineage-specific enhancer upstream of the mouse myeloperoxidase (MPO) gene. Leukemia 8:717, 1994[Medline] [Order article via Infotrieve]
34.
Ford AM,
Bennett CA,
Healy LE,
Towatari M,
Greaves MF,
Enver T:
Regulation of the myeloperoxidase enhancer binding proteins Pu.1, C-EBP-alpha, -beta and -delta during granulocyte-lineage specification.
Proc Natl Acad Sci USA
93:10838,
1996
35.
Sun Z,
Yergeau DA,
Tuypens T,
Tavernier J,
Paul CC,
Baumann MA,
Tenen DG,
Ackermann SJ:
Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene.
J Biol Chem
270:1462,
1995
36.
Yamaguchi Y,
Zhang D-E,
Sun Z,
Albee EA,
Nagata S,
Tenen DG,
Ackermann SJ:
Functional characterization of the promoter for the gene encoding human eosinophil peroxidase.
J Biol Chem
269:19410,
1994 37. Jantscheff P, Grunert F, Nagel G, Thompson J, von Kleist S, Embleton MJ, Price MR, Winter G: A CD66a-specific, activation-dependent epitope detected by recombinant human single chain fragments (scFvs) on CHO-transfectants and activated granulocytes. J Leukoc Biol 59:891, 1996[Abstract]
38.
Schrewe H,
Thompson J,
Bona M,
Hefta LJF,
Maruya A,
Hassauer M,
Shively JE,
von Kleist S,
Zimmermann W:
Cloning of the complete gene for the carcinoembryonic antigen: Analysis of its promotor indicates a region conveying cell type-specific expression.
Mol Cell Biol
10:2738,
1990
39.
Hauck W,
Stanners CP:
Transcriptional regulation of the carcinoembryonic antigene gene. Identification of regulatory elements and multiple nuclear factors.
J Biol Chem
270:3602,
1995 40. Hauck W, Nedellec P, Turbide C, Stanners CP, Barnett TR, Beauchemin N: Transcriptional control of the human biliary glycoprotein gene, a CEA gene family member down-regulated in colorectal carcinomas. Eur J Biochem 223:529, 1994[Medline] [Order article via Infotrieve]
41.
Lei K-J,
Wang C,
Chamberlin ME,
Liu J-L,
Pan C-J,
Chou JY:
Characterization of two allelic variants of a human pregnancy-specific glycoprotein gene.
J Biol Chem
268:17528,
1993 42. Koritschoner NP, Panzetta-Dutari G, Bocco JL, Dumur CI, Flury A, Patrito LC: Analyses of cis-acting and trans-acting elements that are crucial to sustain pregnancy-specific glycoprotein gene expression in different cell types. Eur J Biochem 236:365, 1996[Medline] [Order article via Infotrieve]
43.
Chamberlin ME,
Lei K-J,
Chou JY:
Subtle differences in human pregnancy-specific glycoprotein gene promoters allow for differential expression.
J Biol Chem
269:17152,
1994 44. Kaufman MH: The Atlas of Mouse Development. London, UK, Academic, 1992
45.
Fennie C,
Cheng J,
Dowbenko D,
Young P,
Lasky LA:
CD34+ endothelial cell lines derived from murine yolk sac induce the proliferation and differentiation of yolk sac CD34+ hematopoietic progenitors.
Blood
86:4454,
1995 46. Kale SA, Rao SG: Haematopoietic stem cells during development of mouse embryo. Indian J Exp Biol 30:371, 1992[Medline] [Order article via Infotrieve] 47. Torubarova NA, Dubrovina IV, Zharov VN, Semenova LA, Kopyl'tsova EA, Kudriashova VF, Blidchenko IuA, Koshel' IV, Sukhikh GT: Hematopoiteic cells of fetal liver. 1. Effect of growth factors of varying origin and activity of granulocyte-macrophage progenitor cells. Gematol Transfuziol 40:3, 1995 48. Hestdal K, Ruscetti FW, Ihle JN, Jacobsen SEW, Dubois CM, Kopp WC, Longo DL, Keller JR: Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol 147:22, 1991[Abstract]
49.
Harrison DE,
Astle CM:
Loss of stem cell repopulating ability upon transplantation.
J Exp Med
156:1767,
1982 50. Sigounas G, MacVittie TJ: Transgenic marrow transplantation: A new in vivo and in vitro system for experimental hemopoiesis and radiobiology which employs sequential molecular monitoring of multiple genetic markers. Radiat Res 135:206, 1993[Medline] [Order article via Infotrieve] 51. Delapuerta R, Martinez E, Bravo L, Ahumada MC: Effect of silymarin on different acute inflammation models and on leukocyte migration. J Pharm Pharmacol 48:968, 1996[Medline] [Order article via Infotrieve] 52. Paya M, Ferrandiz ML, Erradi F, Terencio MC, Kijjoa A, Pinto MMM, Alcaraz MJ: Inhibition of inflammatory responses by a series of novel dolabrane derivatives. Eur J Pharmacol 312:97, 1996[Medline] [Order article via Infotrieve] 53. Sakai A: Protection against septic shock in mice with SJC13, an azaindolidine derivative that is a cell adhesion molecule inhibitor. Inflamm Res 45:448, 1996[Medline] [Order article via Infotrieve]
© 1998 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. Yoon, A. Terada, and H. Kita CD66b Regulates Adhesion and Activation of Human Eosinophils J. Immunol., December 15, 2007; 179(12): 8454 - 8462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Payne, G. Huang, E. Sahakian, J. Y. Zhu, N. S. Barteneva, L. W. Barsky, M. A. Payne, and G. M. Crooks Ikaros Isoform X Is Selectively Expressed in Myeloid Differentiation J. Immunol., March 15, 2003; 170(6): 3091 - 3098. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 1998 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Abstract
Introduction
Abstract
-32P] adenosine triphosphate (ATP) and the primer
extension reaction was performed using avian myeloblastosis virus
(AMV) reverse transcriptase in the presence
of 2 U RNasin.
-actin probe (1.2 kb PstI
fragment from plasmid pAL41) was also hybridized.
