![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, November 2006, p. 7953-7965, Vol. 26, No. 21
0270-7306/06/$08.00+0 doi:10.1128/MCB.00718-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance, and JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba 305-8577, Japan
Received 26 April 2006/ Returned for modification 5 June 2006/ Accepted 17 August 2006
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this regard, it is interesting that several lines of c-myb knockdown mutant mice were recently reported, providing important insights into c-Myb function. Whereas the c-myb knockout mutation is embryo lethal, mice with c-myb knockdown (i.e., reduced expression) are born alive, which enables us to perform various analyses in the adult stage. The first example is the c-myb knockdown mouse that exhibits increased megakaryocyte production but diminished production of erythroid and lymphoid cells (4). The second case is that of mice bearing mutations in the KIX domain of p300, which is an interface of p300 interacting with c-Myb. This line of mice displays anemia, thrombocytosis, megakaryocytosis, B-cell deficiency, and thymic hypoplasia (11). In a large-scale suppressor screen with N-ethyl-N-nitrosourea (ENU) aiming to identify mutations capable of ameliorating thrombocytopenia in Mpl-null mice, two lines of c-Myb-dysfunctional mice harboring point mutations individually in the DNA-binding domain and leucine zipper domain were identified (3). Homozygotes of one of these mutant mouse lines displayed multilineage hematopoietic alterations, including anemia, lymphopenia, and eosinopenia, as well as elevated numbers of megakaryocytes, platelets, and hematopoietic stem cells (28). These lines of evidence suggest that c-Myb plays an important role in the erythroid and megakaryocytic differentiation of hematopoietic progenitors, but the precise mechanisms of regulation of expression of the c-myb gene, as well as the stage at which c-Myb functions, remain to be clarified.
Insertion mutagenesis is an inherent event in transgenic mouse studies and is relatively common (19). Approximately 5% of established transgenic mouse lines are found to carry insertional mutations (19). Conversely, an insertion mutagenesis screening procedure is a powerful technique for studying mammalian genome function. Analysis of transgene-induced insertional mutations has successfully led to the identification of new genes and new functions of known and unknown genes (37).
In this study we identified a transgene insertion mutation in proximity to the c-myb gene in one of the transgenic mouse lines harboring the erythropoietin receptor (EPOR) transgene. Homozygotes of this line suffered from anemia, thrombocythemia, and splenomegaly, but these hematological abnormalities were not linked directly to the transgenic expression of EPOR. The transgene insertion appeared instead to block the function of an important enhancer for c-myb gene expression. We found that the transgene is integrated approximately 77 kb upstream of the c-myb gene and disrupts an enhancer that directs c-myb gene expression in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs). The lack of c-Myb in MEPs perturbs the bifurcation of megakaryocyte-erythrocyte differentiation, which results in an increase in megakaryocyte levels and a decrease in erythroid cell levels. These results strongly suggest that c-Myb is an important regulator for the lineage specification of MEPs along either an erythroid or a megakaryocytic pathway.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Flow cytometry. Mononuclear cells (MNCs) from bone marrow (BM-MNCs) or spleen were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD71, anti-CD41, anti-Gr-1, or anti-CD4, phycoerythrin-conjugated anti-Ter119, anti-CD61, anti-CD11b (Mac-1), or anti-CD8, and allophycocyanin-conjugated anti-c-Kit antibodies. These antibodies were from Becton Dickinson Co. (BD). The samples were subjected to flow cytometry using a FACSCalibur (BD), and data were analyzed with the CellQuest program (BD).
Serum thrombopoietin levels and megakaryocyte ploidy. Serum thrombopoietin (TPO) concentrations were measured using sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (22). A double-staining technique and flow cytometry were used to measure megakaryocyte ploidy (35). Briefly, megakaryocytes were identified after labeling with an FITC-anti-CD41 antibody, and BM-MNCs were fixed with 70% ethanol for 30 min on ice. Cells were then washed with phosphate-buffered saline containing 2% bovine serum albumin and incubated for 30 min at 37°C with propidium iodide (50 µg/ml; Sigma). DNA contents were analyzed with a FACS Calibur.
In vitro progenitor assays. Erythroid colony assays were performed by standard procedures (24). For the CFU-erythroid (CFU-E) assays, 1 x 105 BM-MNCs were cultured in methylcellulose medium (MethoCult M3231; StemCell Technologies) containing 1 U/ml recombinant human EPO (generous gift from Chugai Pharmaceutical). After 3 days of culturing, cells were stained with benzidine, and positive colonies were counted as CFU-E. For counting the burst-forming unit (BFU)-E-derived colonies, 2 x 104 MNCs were cultured with 2 U/ml EPO and 100 ng/ml of stem cell factor (SCF; R&D Systems) for 7 days. For the CFU-megakaryocyte (CFU-Meg) assay, 1 x 105 MNCs were cultured with a MegaCult-C kit (StemCell Technologies) in the presence of 50 ng/ml TPO (generous gift from Kirin Brewery), 10 ng/ml interleukin 3 (IL-3; Kirin), 20 ng/ml IL-6 (Kirin), and 50 ng/ml IL-11 (R&D Systems). After 8 days, the colonies were stained for acetylcholine esterase (AchE) as described previously (8), and the number of AchE-positive colonies was counted.
Immunohistochemical staining. Spleens were fixed in 4% paraformaldehyde, followed by embedding in Tissue-Tek OCT compound (Sakura Finetechnical), and were quickly frozen. Cryosections were stained with hematoxylin-eosin (HE) for histological examination and with AchE, anti-B220, and anti-CD3 antibodies as described previously (20).
Identification of the transgene insertion site. Genomic DNA (200 µg) from homozygously transgenic mouse liver was digested with EcoRI. The DNA fragment containing the junction of genomic and transgenic DNA was identified by Southern blot hybridization analysis using the PstI-EcoRI fragment of G1-HRD as a probe. The DNA fraction containing the junction DNA was enriched by using sucrose gradient ultracentrifugation and Southern blot hybridization with the same probe. A genomic DNA bookshelf library (i.e., a mini-genomic DNA phage library) was constructed by using the ZAP Express vector (Stratagene). Isolated DNA fragments were ligated into the ZAP Express vector and packaged with Gigapack III Gold packaging extract (Stratagene). The bookshelf library containing 107 independent PFU was screened using the same probe as that used in the Southern blot analysis. Plasmid clones were generated by the in vivo excision of the positive phage clones following the manufacturer's protocol. The inserts in these ZAP plasmid clones were sequenced with the sequencing primers T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5'-GTAATACGACTCACTATAGGGC-3'). A BLAST search of the DNA sequence utilized the Celera Discovery system.
c-Myb expression in hematopoietic cells. Total RNA (20 µg) extracted from bone marrow, spleen, and liver was electrophoretically separated. The RNA-blotted membrane was hybridized with radiolabeled probes corresponding to the genes c-myb, G3PDH, and Hbs-1 like. Ter119-, CD41-, CD19-, and CD3-positive cells were sorted using FACS Vantage (BD), and total RNA was extracted from sorted cells using an RNeasy Mini kit (QIAGEN). Reverse transcriptase PCR amplification of c-Myb mRNA was performed as previously described (16). For the immunoblotting analysis, BM-MNCs not expressing Mac-1, Gr-1, CD4, or CD8 were prepared by negative selection using a Vario magnetic cell-sorting (MACS) separator (Miltenyi Biotec), and 1 µg/ml anti-c-Myb antibody (clone 1-1; Upstate) was used to detect c-Myb.
MEP cell assay.
MEPs were sorted using FACS Vantage
by the previously described method
(1) with minor
modifications. Briefly, OP9 stromal cells were maintained in
-modified minimum essential medium (Gibco) supplemented with
20% fetal bovine serum (FBS; Gibco). MEPs (1,500 to 2,000 cells/well)
were transferred onto the confluent OP9 stromal cells and cultured in
the presence of 10 ng/ml SCF, 40 ng/ml Flt-3 ligand (R&D Systems),
20 ng/ml IL-11, 20 ng/ml IL-3, 10 ng/ml granulocyte-macrophage
colony-stimulating factor, 10 ng/ml TPO, and 2 U/ml EPO. After 4 days
of culture, MEPs were analyzed by flow cytometry and colony formation
assays. Colonies were counted after cells were stained with benzidine
(erythroid colonies) or AchE (megakaryocyte
colonies).
Construction of retroviral vectors. Murine stem cell virus-internal ribosomal entry site-enhanced green fluorescent protein (MSCV-IRES-GFP) was kindly provided by A. Kume (Jichi Medical University). Murine c-Myb cDNA was ligated into the NotI site of MSCV-IRES-GFP to produce MSCV-c-myb-IRES-GFP. Phoenix-Eco packaging cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% FBS. Phoenix-Eco cells on 6-cm dishes were transfected with 1 µg DNA using a FuGENE transfection kit (Roche). To establish retroviral packaging cell lines, the supernatants from transfected Phoenix-Eco cells were harvested and used to infect dual-potential PT67 cells (Clontech) in the presence of 8 µg/ml Polybrene (Sigma). After 2 to 4 days, GFP-expressing cells were sorted using FACS Vantage and expanded.
Infection of MEPs with retroviral constructs. Retroviral supernatants were collected at 72 h and used to infect MEPs by Magnetofection (OZ Biosciences) as described elsewhere (27). Briefly, sorted MEP cells were placed on OP9 cells for 1 h at 37°C in a 5% CO2 incubator. Retroviral supernatant and CombiMag (3 µl CombiMag/ml supernatant; OZ Biosciences) were incubated for 20 min at room temperature. The resulting mixture was added to MEPs, and the cell culture plate was placed on a magnetic plate for 20 min at 37°C in a 5% CO2 incubator. After the magnetic plate was removed, a dose of cytokines equal to that used in the MEP and OP9 coculture system was added to the cells. After 4 days, cultured MEPs were analyzed by flow cytometry, and benzidine- or AchE-stained colonies were counted.
Bone marrow transplantation (BMT). Transgenic mutant lines of mice were backcrossed with C57BL/6J-Ly-5.2 mice for six generations, and peripheral blood leukocytes obtained from the mice were detected with anti-mouse CD45.2 antibody. At this stage, the backcrossed mice were considered Ly5.2 mice. Eight- to 12-week-old recipient mice (C57BL/6J-Ly-5.1) were irradiated with a single lethal dose (9.2 Gy) and injected with 1 x 106 freshly isolated bone marrow cells obtained from the Ly5.2 homozygously transgenic mice. To assess the reconstitution of bone marrow, peripheral blood or bone marrow cells were analyzed for chimerism, expressed as the percentage of Ly5.2-expressed leukocytes determined by using anti-Ly5.2 antibody (BD).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
25% of the offspring from line B showed anemia,
thrombocythemia, and splenomegaly, while homozygotes from lines A and C
never showed such abnormalities. We genotyped the progeny of several
crossings of line B heterozygous mice by Southern blotting, and the
results demonstrated that the hematopoietic abnormalities were observed
only in the mice harboring the transgene homozygously (Fig.
1B). These abnormalities
were absent in heterozygous or nontransgenic animals from the same
litter.
|
To further investigate the influence of the genetic background, heterozygously transgenic mice were bred with C57BL/6 mice for eight generations, and then homozygously transgenic mice were generated by intercrossing of these heterozygously transgenic mice. The homozygously transgenic mice with the C57BL/6 background also exhibited anemia, thrombocythemia, and splenomegaly, consistent with the abnormality observed with the BDF1 background (data not shown). This result supports our contention that insertion of the transgene disrupted a gene essential for erythroid and megakaryocytic differentiation.
Hematological analysis of homozygously transgenic mice. The significant traits of the homozygous line B mice are anemia and thrombocythemia. The hemoglobin (Hb) and hematocrit (Ht) levels of the homozygously transgenic mice were significantly lower than those of the heterozygously transgenic and wild-type mice (Table 1). In contrast, platelet counts in homozygotes were approximately three times higher than those in heterozygotes and wild-type mice. White blood cell counts did not differ significantly among these three genotypes.
|
Identification of transgenic insertion sites. To determine the chromosomal localization of the transgene, we prepared a genomic DNA library utilizing EcoRI DNA fragments from a line B homozygously transgenic mouse. To this end, we performed a series of sucrose density gradient centrifugation and Southern blotting analyses with the G1-HRD-EPOR fragment as a probe and isolated a genomic DNA fraction enriched with the portion containing the junction of genomic and transgene DNA. This genomic DNA fraction was ligated into a lambda phage ZAP Express vector to generate a mini-genomic DNA library (or a genomic DNA bookshelf). We subsequently screened the bookshelf using the same G1-HRD-EPOR probe and isolated positive clones, with several phage clones being analyzed by standard procedures. An insertion site was finally identified by sequencing the genomic DNA clones and comparing the sequence with the transgene and mouse genome sequences obtained from the Celera Discovery system.
The transgene insertion site was localized on mouse chromosome 10qA3 (Fig. 2A), which corresponds to human chromosome 6q22-23. Although we could not find a gene directly interrupted by the transgene insertion (Fig. 2B to D), there are three known genes, c-myb, G3PDH pseudo, and Hbs-1 like, neighboring the integration site. We also found that the EST clone BF720900 mapped near the insertion site.
|
To further examine the expression level of the c-Myb protein in homozygously transgenic mice, we enriched erythroid and megakaryocytic cells from BM-MNCs by depleting Mac-1+, Gr-1+, CD4+, and CD8+ cells using the MACS system (see Materials and Methods). Enriched BM-MNCs from homozygously transgenic mice were found to express c-Myb at approximately 15% of the level seen in an equivalent fraction from wild-type mice (Fig. 2G). These data indicate that insertion of the transgene 77 kb upstream of the c-myb gene affects the c-myb gene expression in erythroid- and megakaryocytic-lineage cells and suggest that the transgene insertion site is important for lineage-specific expression of the c-myb gene.
Homozygously transgenic mice exhibit defects in erythroid differentiation. To determine how erythroid differentiation was blocked in homozygously transgenic mice, we examined the status of erythropoiesis. Erythroid early progenitors at the BFU-E stage were significantly diminished in the bone marrow of homozygously transgenic mice (Fig. 3A). The BFU-E colonies from homozygously transgenic mice were significantly smaller than those from wild-type and heterozygously transgenic mice (Fig. 3A, inset). CFU-E colony numbers were decreased significantly in homozygously transgenic mice (Fig. 3B). These results indicate that erythroid differentiation was blocked in homozygous line B mice at or before the BFU-E stage.
|
Increase of megakaryocytes and platelets in transgene homozygotes. Megakaryopoiesis in the bone marrow of line B homozygously transgenic mice was also examined. In agreement with the observation that megakaryocytes were increased in homozygously transgenic mouse bone marrow (Fig. 1H), the numbers of CFU-Meg colonies obtained with cells from these mice were approximately threefold higher than those from wild-type and heterozygously transgenic mice (Fig. 4A). In contrast, the serum concentration of TPO, a cytokine stimulator of megakaryopoiesis, was not elevated in homozygously transgenic mice compared with wild-type and heterozygously transgenic mice (Fig. 4B). These findings imply that the increased megakaryopoiesis and platelet production were caused by a mechanism independent of the TPO pathway.
|
Homozygously transgenic mice showed splenomegaly with megakaryocytosis. The spleens of homozygously transgenic mice were markedly enlarged compared with those of wild-type and heterozygously transgenic mice (Fig. 5A). The total cell counts in spleens from homozygously transgenic mice were three times ([1.3 ± 0.1] x 108) higher than those in wild-type mice ([4.3 ± 0.4] x 107). Histological examination of homozygous spleens revealed that the red pulp was significantly enlarged and the structure of the white pulp was disrupted (Fig. 5B to D). The number of megakaryocytes was significantly increased in the spleens of homozygously transgenic mice and were detectible as large cells (Fig. 5E to G) or AchE-staining cells in enlarged red pulps (Fig. 5H to J). Whereas the white pulp of spleen was filled with B220-positive B cells in wild-type and heterozygously transgenic mice, in homozygously transgenic mice the white pulp was disorganized and numbers of B220-positive cells were reduced (Fig. 5K to M). T-cell-marker-positive cells were relatively unchanged; the cells make up approximately 20% of the wild-type, heterozygously transgenic, and homozygously transgenic mouse spleen cells. Myeloid-marker-positive-cell levels were also similar in wild-type, heterozygously transgenic, and homozygously transgenic mouse spleen.
|
Megakaryocyte-erythroid common progenitor cells are affected in homozygously transgenic mice.
To determine the
mechanisms underlying the erythroid-megakaryocytic differentiation
imbalance, we examined MEPs in the homozygously transgenic mice. MEPs
are characterized as Fc
Rlow
CD34+ c-Kit+
Sca-1– IL-7R–
Lin– cells, and when cultured in the presence of EPO
and TPO, MEPs give rise to CFU-Meg, BFU-E, and CFU-E colonies
(1). We found that c-Myb
mRNA expression was significantly diminished in the MEPs of
homozygously transgenic mice compared with that in cells of wild-type
mice (Fig.
6A).
|
Retroviral complementation of c-Myb rescued mutant MEPs from abnormal differentiation. To clarify whether the reduction of c-Myb in MEPs was the direct cause of the hematopoietic abnormalities, we introduced c-Myb retrovirally into the mutant MEPs. For this purpose, mouse c-Myb cDNA was inserted into the MSCV-IRES-GFP vector, and this c-Myb-expressing virus was used to infect MEPs isolated from the homozygously transgenic or wild-type mice. Since the c-Myb virus-infected cells emit a GFP signal, these cells were easily identified by fluorescent microscopy (Fig. 7A). We found an increase in the frequency of benzidine-positive erythroid cells in c-Myb virus-infected mutant MEPs, but no such increase was observed in the control vector-infected cells (Fig. 7B).In contrast, the frequency of AchE-positive megakaryocytes was decreased in the c-Myb virus-infected mutant MEPs (Fig. 7C). This indicates that the mutant MEP from homozygously transgenic mice can reproduce in vitro the hematological abnormalities observed in homozygously transgenic mice in vivo. Furthermore, retroviral complementation of c-Myb effectively rescued the mutant MEPs from the differentiation imbalance. These data indicate that the transgene insertion upstream of the c-myb gene affects hematopoietic cells especially at the stage of MEP and that the most essential function of c-Myb for the bifurcation of erythroid and megakaryocyte lineages exists during the stage of MEPs.
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
In this regard, it should be noted that there are a few previous reports of cases in which transgene insertion into an important regulatory region indeed caused perturbation of gene expression. One salient example of such cases is doubleridge, a transgene-induced mutation resulting in mice displaying forelimb postaxial polysyndactyly. The transgene insertion site of doubleridge was identified as 150 kb downstream of the dickkopf1 (Dkk1) gene, and because of the transgene integration, the expression level of Dkk1 was reduced to less than 1% of the wild-type level in E13.5 mutant embryos (15).
As mentioned above, changes in c-Myb mRNA and protein expression were marginal when we examined total MNCs in the bone marrow and spleen. c-Myb mRNA expression was reduced specifically in the MEPs, Ter119+ erythroid cells, and CD41+ megakaryocytes. Thus, in the present case the transgene insertion perturbed c-myb gene expression in a lineage-specific manner. In contrast, the expression level or activity of c-Myb was reduced fairly uniformly in the other recently reported c-myb knockdown mutant mouse cases (3, 4, 11). While the mechanisms leading to reduced c-myb gene expression differ among these mutant animals, the resulting phenotypes are similar, namely anemia, thrombocythemia, B-cell deficiency, and maturation impairment of T cells.
In line B homozygously transgenic mice, while B cells were depleted, thymus development and T-cell maturation were normal. We therefore carried out preliminary c-Myb expression analysis in lymphoid lineages and found that c-Myb mRNA expression was decreased in the B-cell fraction but increased in the T-cell fraction. Thus, the c-Myb expression in each hematological lineage correlated with the abnormalities in the mutant mice. These results suggest that there may be a T-cell-oriented regulatory domain in the c-myb gene, which is independent of the one currently disrupted with the transgene integration. Since normal T-cell maturation was not observed in the other c-myb knockdown mutant mouse cases, our mutant mouse line provides a unique mechanism for studying c-Myb reduction and phenotype in the lymphoid lineages as well.
Although line B homozygously transgenic mice suffer from anemia, thrombocytosis, and splenomegaly, these mice lived approximately 2 years in our specific-pathogen-free animal facility, showing a life span similar to that of wild-type mice (data not shown). In this long-term observation study, aged line B homozygously transgenic mice suffered from splenic fibrosis, but we did not observe significant occurrence of specific malignancies, such as lymphoma (data not shown). These data indicate that the transgene integration gives rise to perturbation in c-myb gene expression in line B mice, but the perturbation was not so serious as to affect the life span of the mice.
In the classical model of hematopoiesis, erythroid and megakaryocyte lineages arise from MEPs derived from common myeloid progenitors (1, 10). Although erythroid and megakaryocyte lineages are believed to share the common progenitors (1, 10), the mechanism regulating the final separation of these two lineages is not well defined. During hematopoietic differentiation from lineage commitment to terminal maturation, a multiple complex of transcription factors coordinately regulates the chromatin organization of lineage-specific genes and primes them for expression (29). Transcription factors whose targeted disruptions lead primarily to defects in the megakaryocyte and erythroid lineages, such as GATA-1 (26, 30) and NF-E2 p45 (13), are highly expressed in MEPs (1), and these transcription factors positively promote both erythroid and megakaryocytic lineages. In contrast, c-Myb has opposite effects on erythroid and megakaryocytic lineages; the present study clearly indicates that c-Myb is required to promote erythroid lineage proliferation and differentiation while suppressing megakaryopoiesis. This contrasting effect is a unique function of c-Myb in the development of erythroid and megakaryocyte lineages.
The erythroid/megakaryocytic abnormalities were reconstituted in vivo in sublethally irradiated mice through the transplantation of mutant mouse bone marrow cells, demonstrating that the hematological abnormalities of the mutant mice are cell autonomous. The coculture experiment of MEPs with OP9 stromal cells successfully reproduced the blockage of erythroid differentiation and promotion of megakaryocyte differentiation in vitro, further supporting this notion. A twofold reduction in the abundance of MEPs seen in our mutant mice was also found in mice with an ENU-induced c-Myb knockdown mutation (c-MybM303V/M303V) (28). Importantly, while levels of hematopoietic stem cells were increased 5- to 10-fold in c-MybM303V/M303V mice (28), those in our c-Myb mutant mice were unchanged or slightly decreased compared to those in wild-type mice (data not shown). These results support our contention that c-Myb contributes to the bifurcation of erythroid and megakaryocytic lineages during the stage of MEP. We surmise that further analysis of the c-Myb function in MEPs will clarify molecular mechanisms of erythroid-megakaryocyte divergence through MEPs.
|
ACKNOWLEDGMENTS |
|---|
This work was supported in part by grants from JST-ERATO (M.Y.) and the Ministry of Education, Culture, Sports, Science and Technology (H.Y.M., H.M., and M.Y.).
|
FOOTNOTES |
|---|
Published
ahead of print on 28 August 2006.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Barr, N. I., M. Stewart, C. Tsatsanis, R. Fulton, M. Hu, H. Tsujimoto, and J. C. Neil. 1999. The fit-1 common integration locus in human and mouse is closely linked to MYB. Mamm. Genome 10:556-559.[CrossRef][Medline]
3. Carpinelli,
M. R., D. J. Hilton, D. Metcalf, J. L.
Antonchuk, C. D. Hyland, S. L. Mifsud, L.
D. Rago, A. A. Hilton, T. A. Willson, A.
W. Roberts, R. G. Ramsay, N. A. Nicola, and
W. S. Alexander. 2004. Suppressor screen in
Mpl–/– mice:
c-Myb mutation causes supraphysiological production of
platelets in the absence of thrombopoietin signaling. Proc.
Natl. Acad. Sci. USA
101:6553-6558.
4. Emambokus, N., A. Vegiopolus, B. Harman, E. Jenkinson, G. Anderson, and J. Frampton. 2003. Progression through key stage of hematopoiesis is dependent on distinct threshold levels of c-Myb.EMBO J. 22:4478-4488.[CrossRef][Medline]
5. Friedman, A. D. 2002. Runx1, c-Myb, and C/EBP alpha couple differentiation to proliferation or growth arrest during hematopoiesis. J. Cell. Biochem. 86:624-629.[CrossRef][Medline]
6. Hanlon, L., N. I. Barr, K. Blyth, M. Stewart, P. Haviernik, L. Wolff, K. Weston, E. R. Cameron, and J. C. Neil.2003 . Long-range effects of retroviral insertion on c-mybi: overexpression may be obscured by silencing during tumor growth in vitro. J. Virol. 77:1059-1068.[Medline]
7. Haviernik,
P., S. M. Festin, R. Opavsky, P. P. Koller,
N. I. Barr, J. C. Neil, and L. Wolff.2002
. Linkage on chromosome 10 of several murine
retroviral integration loci associated with leukaemia.J. Gen. Virol.
83:819-827.
8. Jackson,
C. W. 1973. Cholinesterase as a possible
marker for early cells of the megakaryocytic series.Blood
42:413-42122.
9. Jiang, X., L. Villeneuve, C. Turmel, C. A. Kozak, and P. Jolicoeur. 1994. The Myb and Ahi-1 genes are physically very closely linked on mouse chromosome 10.Mamm. Genome 5:142-148.[CrossRef][Medline]
10. Kanz, L., G. Straub, K. G. Bross, and A. A. Fauser.1982 . Identification of human megakaryocytes derived from pure megakaryocytic colonies (CFU-M), megakaryocytic-erythroid colonies (CFU-M/E), and mixed hemopoietic colonies (CFU-GEMM) by antibodies against platelet associated antigens. Blut 45:267-274.[CrossRef][Medline]
11. Kaper, L. H., F. Boussouar, P. A. Ney, C. W. Jackson, J. Rehg, J. M. van Deursen, and P. K. Brindle. 2002. A transcription-factor-binding surface of coactivator p300 is required for hematopoiesis.Nature 419:738-743.[CrossRef][Medline]
12. Kastan,
M. B., D. J. Slamon, and C. I. Civin.1989
. Expression of protooncogene c-myb in normal
human haemopoietic cells. Blood
73:1444-1451.
13. Levin,
J., J.-P. Peng, G. R. Baker, J.-L. Villeval, P. Lecine,
S. A. Burstein, and R. A. Shivdasani.1999
. Pathophysiology of thrombocytopenia and anemia in
mice lacking transcription factor NF-E2. Blood
94:3037-3047.
14. Longmore,
G. D., P. Pharr, D. Neumann, and H. F. Lodish.1993
. Both megakaryocytopoiesis and erythropoiesis are
induced in mice infected with a retrovirus expressing an oncogenic
erythropoietin receptor. Blood
82:2386-2395.
15. MacDonald,
B. T., M. Adamska, and M. H. Meisler.2004
. Hypomorphic expression of Dkk1 in the
doubleridge mouse: dose dependence and compensatory
interactions with Lrp6. Development
131:2543-2552.
16. McClanahan,
T., S. Dalrymple, M. Barkett, and F. Lee. 1993.
Hematopoietic growth factor receptor genes as markers of lineage
commitment during in vitro development of hematopoietic cells.Blood
81:2903-2915.
17. McDonald, T. P., M. B. Cottrell, R. E. Clift, W. C. Cullen, and F.-K. Lin. 1987. High doses of recombinant erythropoietin stimulate platelet production in mice. Exp. Hematol. 15:719-721.[Medline]
18. Miller,
C. P., Z. Y. Liu, C. T. Noguchi, and
D. M. Wojchowski. 1999. A minimal
cytoplasmic subdomain of the erythropoietin receptor mediates erythroid
and megakaryocyte cell development. Blood
94:3381-3387.
19. Misler, M. H. 1992. Insertional mutation of ‘classical’ and novel genes in transgenic mice.Trends Genet. 8:341-344.[Medline]
20. Morito,
N., K. Yoh, Y. Fujioka, T. Nakano, H. Shimohata, Y. Hashimoto,
A. Yamada, A. Maeda, F. Matsuno, H. Hata, A. Suzuki, S.
Imagawa, H. Mitsuya, H. Esumi, A. Koyama, M. Yamamoto, N.
Mori, and S. Takahashi. 2006. Overexpression of c-Maf
contributes to T-cell lymphoma in both mice and human. Cancer
Res.
66:812-819.
21. Mucenski, M. L., K. McLain, A. B. Kier, S. H. Swederlow, C. M. Schreiner, T. A. Miller, D. W. Piertryga, W. J. Scott, Jr., and S. S. Potter. 1991. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.Cell 65:677-689.[CrossRef][Medline]
22. Nishiyama, U., H. Morita, Y. Torii, T. Kuwaki, E. Shimizu, H. Akahori, T. Kato, and H. Miyazaki. 2001. Platelets exposed to elevated levels of endogenous thrombopoietin in vivo have a reduced response to megakaryocyte growth and development factor in vitro. Thromb. Haemost. 85:152-159.[Medline]
23. Oh, I. H., and E. P. Reddy. 1999. The myb gene family in cell growth, differentiation and apoptosis.Oncogene 18:3017-3033.[CrossRef][Medline]
24. Ohneda,
O., N. Yanai, and M. Obinata. 1990. Microenvironment
created by stromal cells essential for a rapid expansion of erythroid
cells in mouse fetal liver. Development
110:379-384.
25. Onodera, K., J. A. Shavit, H. Motohashi, M. Yamamoto, and J. D. Engel. 2000. Perinatal synthetic lethality and hematopoietic defects in compound mafG::mafK mutant mice. EMBO J. 19:1335-1345.[CrossRef][Medline]
26. Pevny, L., M. C. Simon, E. Robertson, W. H. Klein, S. F. Tsai, V. D'Agati, S. H. Orkin, and F. Costantini. 1991. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349:257-260.[CrossRef][Medline]
27. Plank, C., U. Schillinger, F. Scherer, C. Bergemann, J.-S. Remy, F. Krotz, M. Anton, J. Lausier, and J. Rosenecker. 2003. The Magnetofection method: using magnetic force to enhance gene delivery.Biol. Chem. 384:737-747.[CrossRef][Medline]
28. Sandberg, M. L., S. E. Sutton, M. T. Pletcher, T. Wiltshire, L. M. Tarantino, J. B. Hogensch, and M. P. Cooke. 2005. c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation. Dev. Cell 8:153-166.[CrossRef][Medline]
29. Shivdasani,
R. A., and S. H. Orkin. 1996. The
transcriptional control of hematopoiesis. Blood
87:4025-4039.
30. Shivdasani, R. A., Y. Fujiwara, M. A. McDevitt, and S. H. Orkin. 1997. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 16:3965-3973.[CrossRef][Medline]
31. Socolovsky,
M., H-S. Nam, M. D. Fleming, V. H. Haase, C.
Burgnara, and H. F. Lodish. 2001.
Ineffective erythropoiesis in Stat5a-/-5b-/-mice due to decreased
survival of early erythroblastosis. Blood
98:3261-3273.
32. Sumner, R., A. Crawford, M. Mucenski, and J. Frampton. 2000. Initiation of adult myelopoiesis can occur in the absence of c-Myb whereas subsequent development is strictly dependent on the transcription factor. Oncogene 19:3335-3352.[CrossRef][Medline]
33. Suzuki,
N., O. Ohneda, S. Takahashi, M. Higuchi, H. Y. Mukai, T.
Nakahata, S. Imagawa, and M. Yamamoto. 2002.
Erythroid-specific expression of the erythropoietin receptor rescued
its null mutant mice from lethality. Blood
100:2279-2288.
34. Suzuki,
N., N. Suwabe, O. Ohneda, N. Obara, S. Imagawa, X. Pan, H. Motohashi,
and M. Yamamoto. 2003. Identification and
characterization of 2 types of erythroid progenitors that express
GATA-1 at distinct levels. Blood
102:3575-3583.
35. Toki,
T., F. Katsuoka, R. Kanezaki, G. Xu, H. Kurotaki, J. Sun, T. Kamio, S.
Watanabe, S. Tandai, K. Terui, S. Yagihashi, N. Komatsu, K. Igarashi,
M. Yamamoto, and E. Ito. 2005. Transgene expression of
BACH1 transcription factor results in megakaryocytic impairment.Blood
105:3100-3108.
36. Wolff, L. 1996. Myb-induced transformation. Crit. Rev. Oncog. 7:245-260.[Medline]
37. Woychik, R. P., and K. Alagramam. 1998. Insertional mutagenesis in transgenic mice generated by the pronuclear microinjection procedure. Int. J. Dev. Biol. 42:1009-1017.[Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
