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1 Department of Pathology, and 2 Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, Suita, Osaka 565–0871, Japan
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Abstract |
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Introduction |
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The FOG family is a novel class of multiple zinc finger proteins that interact with GATA factors (Fox et al. 1998, 1999; Cantor et al. 2002; Chang et al. 2002). The founding member of this family, FOG-1, was cloned by yeast two-hybrid screening, using the amino terminal zinc finger of GATA-1 as bait (Tsang et al. 1997). FOG-1 is a polypeptide of 998 amino acids that contains nine zinc fingers; four of the fingers interact independently with GATA-1 (Fox et al. 1999). FOG-1 is expressed abundantly in erythroid and megakaryocytic cells, which correlates with the expression pattern of GATA-1. FOG-1-null mice die of anaemia by embryonic day 11.5 (Tsang et al. 1998). Erythroid cells of the mutant mice show a marked but partial blockage, which is reminiscent of GATA-1-null erythroid precursors. In contrast to erythropoiesis, megakaryocytes do not develop in FOG-1-null mice. Null mutations in FOG-1 exhibit more profound effects on megakaryocytes than on erythrocytes.
The Xenopus laevis homologue of FOG (xFOG) has been cloned, and its structure has been found to be highly similar to FOG-1. Ectopic expression and explant assays have demonstrated that the xFOG protein acts as a repressor of erythroid lineage cells in vivo (Deconinck et al. 2000). In addition, the Drosophila FOG homologue U-shaped acts as a negative regulator of blood cells (Haenlin et al. 1997; Fossett et al. 2001). However, in mammals, FOG-1 over-expression in the erythroid lineage cell line G1E and myeloid cell line 416B promoted erythroid and megakaryocytic differentiation, respectively, when coexpressed with GATA-1 (Tsang et al. 1997; Crispino et al. 1999). This discrepancy between mammals and the other species may be attributable to species differences or to the fact that the data on mammalian erythroid and megakaryocytic differentiation have been derived from established cell lines, which do not necessarily reflect the physiological differentiation process.
In vitro differentiation from mouse embryonic stem (ES) cells is a powerful tool for the analysis of the development and differentiation of various biological systems (Nakano 2003). Two methods have been reported for haematopoietic differentiation in ES cells: (1) embryoid body formation; and (2) coculture with the macrophage-colony stimulating factor (M-CSF)-deficient stroma cell line OP9. We have utilized the OP9 system to analyse various aspects of haematopoietic development and differentiation (Nakano et al. 1996, 1997). The OP9 system recapitulates the early phase of haematopoietic development and the differentiation from multipotential haematopoietic progenitors to all of the myeloid lineage cells, B lineage cells, and NK cells. It is noteworthy that differentiation is induced only once in the OP9 system, and that differentiation proceeds in a highly synchronous manner. Thus, at one distinct time point after the initiation of differentiation induction, most cells belong to a single differentiation stage.
Recently, the tetracycline-based conditional gene expression method has been used successfully with the OP9 system (Era & Witte 2000; Era 2002; Kitajima et al. 2002). The combination of conditional gene expression and OP9 cells enables us to analyse the function of various genes at distinctive differentiation stages of haematopoietic development and differentiation. In the present study, we used this method to analyse the functions of FOG-1 in erythroid cells, megakaryocytic cells, and ES cells. Unexpectedly, the effects of FOG-1 on the different stages of megakaryocyte differentiation varied significantly. Furthermore, experiments using the GATA-1-null haematopoietic cells, or the conditional expression of a mutant form of FOG-1 that could not bind to the transcriptional corepressor protein C-terminal binding protein (CtBP), revealed that different binding proteins were necessary for the distinctive effects of FOG-1. These data clearly show that the functions of FOG-1 should be interpreted in a differentiation context-dependent manner.
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Results |
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In order to understand the biological roles of FOG-1 in stage- and lineage-specific haematopoietic cells, we established ES cell lines that express FOG-1 under the control of the TET-Off conditional gene expression system. The Tet-O CMV-FOG-1-IRES-EGFP was introduced by electroporation into an ES cell line that expressed the Tet-regulatable transcription factor tTA-2 (Fig. 1A). Forty-eight clones were selected, and analysed for conditional regulation of EGFP expression. Eight clones showed EGFP expression that was strictly regulated, i.e. these clones expressed EGFP only in the absence of tetracycline. Clones nos. 20 and 39 were chosen for further analysis, as a high percentage of these cells expressed EGFP (96.5% and 94.3% for clones nos. 20 and 39, respectively) 24 h after tetracycline deprivation (Fig. 1B,C). The expression of FOG-1 was confirmed by Western blotting (Fig. 1D), and the expression levels of induced FOG-1 correlated inversely with the concentrations of tetracycline (Fig. 1E). Since these two clones showed essentially similar results in subsequent experiments, the results for clone no. 39 are presented as representative data.
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In order to define in detail the roles of FOG-1 during haematopoietic differentiation, in vitro haematopoietic differentiation of the ES cells was performed, so that conditional FOG-1 expression could be achieved. As previously described, multipotential haematopoietic progenitors began to appear on day 5 of OP9 in vitro haematopoietic differentiation. At day 7 of induction, ES cells gave rise to immature haematopoietic colonies. Primitive embryonic and definitive adult erytroid cells appeared between days 6 and 8 and between days 12 and 15, respectively, in the presence of eryhtropoietin (EPO). FOG-1 expression during the differentiation from control ES cells in the presence of TPO was examined by RT-PCR analysis (Fig. 1F). FOG-1 was detectable between day 5 and day 11. To reveal the activity of FOG-1 on immature progenitors, FOG-1 was over-expressed from day 5, and the colonies were counted on day 7 (Fig. 2A). The number of haematopoietic colonies decreased to < 20% as the result of FOG-1 over-expression. The development of primitive erythrocytes was analysed on day 8 of differentiation induction in the presence of EPO. The total number of cells was drastically reduced (Fig. 2B), and the vast majority of the cells expressed neither TER-119 nor an earlier erythroid marker, the CD71/transferrin receptor (Fig. 2C). These data show that FOG-1 over-expression impairs the development of immature haematopoietic progenitors and primitive erythrocytes.
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Differentiation stage-dependent effects of FOG-1 on megakaryopoiesis
We also examined the effects of FOG-1 on the proliferation and differentiation of megakaryocytes following the addition of thrombopoietin (TPO). FOG-1 was expressed starting on day 5, and the number of colonies was counted on day 8 postinduction. As shown in Fig. 3A,B, FOG-1 over-expression reduced the numbers of immature megakaryocytic colonies and mature megakaryocytes on days 8 and 12, respectively. Compared to the effects of FOG-1 on erythroid cells (Fig. 2A,B), those on megakaryocytes were mild. Proliferation of erythroid cells was inhibited by FOG-1 throughout the differentiation, but that of megakaryocytes was enhanced at the late phase of differentiation as shown below. Therefore, at least in part, milder effect of FOG-1 can be explained by the proliferation of megakaryocytes at later phase of differentiation. FOG-1 did not alter significantly the expression pattern of either the megakaryocyte marker platelet glycoprotein V (Fig. 3C) or GPIIb (data not shown), both of which are expressed during the stages from immature to mature megakaryocytes. Thus, FOG-1 expression seems to suppress megakaryopoiesis, as well as erythropoiesis. Then, as in the erythropoiesis experiments, the expression of FOG-1 was induced for restricted periods of time, to allow the analysis of the megakaryocyte differentiation stage-dependent function of FOG-1. Unexpectedly, the number of megakaryocytes increased two-fold when FOG-1 was expressed after day 8 (Fig. 3D).
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The second experiment consisted of a colony formation assay. OP9 cell differentiation was induced until day 8. The induced cells were transferred to methylcellulose semisolid medium that contained TPO, and the numbers of small, medium, and large colonies were assessed. During the course of the experiment, FOG-1 was expressed for the indicated time periods (Table 1). Compared to the control culture, the numbers of small and middle sized colonies decreased significantly when FOG-1 was expressed between days 5 and 8, and between days 5 and 12. In contrast, these numbers were not affected, and the number of large colonies was significantly increased, when FOG-1 was expressed starting on day 8. Therefore, FOG-1 over-expression between days 5 and 8 represses the production of megakaryocyte progenitors from multipotential haematopoietic progenitors, whereas FOG-1 expression after day 8 enhances the proliferation of megakaryocytes.
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The expression patterns of FOG-1, GATA-1, and GATA-2 were analysed after the cells were transferred to the suspension culture condition, starting at day 8. Western blot analysis was carried out to examine the FOG-1 expression levels (Fig. 4A). Endogenous FOG-1 expression decreased after day 8. Although FOG-1 expression was detected on days 10 and 12, even in the presence of tetracycline, expression was higher in the absence of tetracycline. The results of the RT-PCR analysis of FOG-1 correlated well with the Western blot findings (Fig. 4B). The expression of GATA-1 was not affected significantly by FOG-1, while GATA-2 expression was higher in the cells that over-expressed FOG-1.
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Effects of the C-terminal-binding protein on the function of FOG-1
It has been reported that FOG-1 binds to a transcriptional corepressor, CtBP, and that this binding may influence FOG-1 function (Fox et al. 1999; Deconinck et al. 2000; Katz et al. 2002). A FOG-1 mutant that was unable to bind to CtBP (FOG-1
CtBP) was produced, and its effects on erythroid and megakaryocytic differentiation were analysed using the same experimental system described here. The FOG-1CtBP mutation and its conditional expression pattern are shown in Fig. 5A–C. The data on FOG-1CtBP over-expression are quite similar to those obtained for wild-type FOG-1 over-expression (Fig. 5D–G). FOG-1CtBP repressed the production of haematopoietic progenitors and erythroid lineage cells. Although megakaryocyte production was repressed by FOG-1CtBP over-expression from day 5 to day 8, it was enhanced by FOG-1CtBP over-expression from day 8. The phenotypes of the FOG-1CtBP-over-expressing megakaryocytes were identical to those of the FOG-1-over-expressing controls (data not shown).
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CtBP on megakaryopoiesis in GATA-1-null cells
We produced GATA-1-null ES cells and generated Tet-off expression of FOG-1, to examine in detail the co-operative effects of GATA-1 and FOG-1. First, we examined the megakaryocyte development form GATA-1 null ES cells by OP9 system. The number of megakaryocytes was significantly reduced by the null mutation of GATA-1 (Fig. 6A). Differentiation of megakaryocytes was also impaired by the mutation (data not shown). Although megakaryocyte markers such as GPV, GPIIb and acetylcholine-esterase were normally expressed, proplatelet formation was hardly detected. Next, we examined the effects of FOG-1 and FOG-1CtBP under the GATA-1 null condition. When either FOG-1 or FOG-1CtBP was expressed between days 5 and 8, the number of megakaryocytes decreased (Fig. 6B,C). However, FOG-1 or FOG-1CtBP expression after day 8 did not alter the number of megakaryocytes. These data show that whereas the interaction with GATA-1 is indispensable for late-phase enhancement, it is dispensable for early phase inhibition by FOG-1. Although GATA-1 is dispensable for the early phase inhibition, GATA-1 null mutation brought about more profound inhibitory effect of FOG-1. This implies that simple co-operative effects of FOG-1 and GATA-1 cannot explain the inhibition by FOG-1.
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CtBP gave essentially similar results (data not shown). In the GATA-1 KO megakaryocytes, the GATA-2 expression level was similar to that of the control megakaryocytes (Figs 4B and 6J). Therefore, expression level of GATA-2 does not seem to give significant influence on the megakaryocyte proliferation and differentiation. Since GATA-2 can collaborate with FOG-1, we cannot exclude the possibility that FOG-1 would play some role in megakaryocyte differentiation by synegizing with GATA-2. FOG-1 increased the expression of GATA-2 in the GATA-1-null context, but this increase was smaller than that in the control. Another haematopoietic GATA factor, GATA-3, was not expressed in the megakaryocytes under any condition (data not shown). These data show that FOG-1 inhibits megakaryocyte maturation in a GATA-1-independent manner.
Effects of FOG-1 and FOG-1CtBP on ES cell proliferation
We also analysed the effects of FOG-1 on ES cells. ES cell growth was impaired significantly by FOG-1 over-expression. After day 6, the number of cells was reduced to about 25% of the control number (Fig. 7A). The decrease in EGFP suggests poor proliferation of the FOG-1-expressing cells (Fig. 7B). Cell cycle analysis showed that the cells that expressed FOG-1 were arrested at the G0/G1 phase (Fig. 7C). This inhibitory effect of FOG-1 was similarly observed when FOG-1 was over-expressed in the GATA-1 null ES cells (data not shown). The over-expression of FOG-1CtBP produced completely different results. As shown in Fig. 7D, FOG-1CtBP did not affect the ES cell proliferation. Morphological analysis showed no particular difference between the control and FOG-1-over-expressing ES cells (data not shown). Furthermore, the differentiation markers of the three germ layers were not altered by FOG-1 over-expression (data not shown). These data suggest that ES cell differentiation is not induced by FOG-1 over-expression. RT-PCR analysis revealed that none of the GATA factors were expressed in the ES cells (data not shown). Therefore, FOG-1 inhibits the proliferation of ES cells in a CtBP-dependent, GATA-independent manner.
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Discussion |
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When FOG-1 was expressed between days 5 and 8, i.e. from the stage of multipotential or erythro-megakaryocytic progenitors to megakaryocytic progenitors, the proliferation of megakaryocytic cells was impaired. However, during terminal differentiation of the megakaryocytes, FOG-1 enhanced the proliferation and inhibited the maturation of megakaryocytes. An elegant series of experiments and the analysis of GATA-1-mutant patients have revealed that FOG-1 is essential for megakaryopoiesis, and that this effect is dependent on co-operation with GATA-1 (Nichols et al. 2000; Freson et al. 2001, 2002; Mehaffey et al. 2001). Interaction between FOG-1 and GATA-1 would explain the enhanced proliferation of megakaryocytes, since in the GATA-1-null condition, proliferation was not enhanced by FOG-1 over-expression. However, the decreased proliferation of early megakaryocyte progenitors by FOG-1 was GATA-1-independent, since the effect was observed even in the GATA-1-null condition. This inhibitory effect might be explained by the co-operation of FOG-1 with GATA-2.
Considering the effect of GATA-2 on the enhancement of multipotential haematopoietic progenitors (Kitajima et al. 2002) and the phenotype of GATA-2-null mice (Tsai et al. 1994; Shivdasani & Orkin 1996), the reduced proliferation seen during the multipotential progenitor phase may be explained by FOG-1 interference with the transcription of GATA-2. Meanwhile, the increase in GATA-2 might enhance the proliferation of megakaryocytes in the late stage of differentiation, since GATA-2 expression on day 10 was increased by the expression of FOG-1 in the presence of TPO (Fig. 4B). However, since the simple over-expression of GATA-2 from day 8 of differentiation did not induce the proliferation or differentiation block of megakaryocytes (unpublished data), the increased expression of GATA-2 alone cannot account for the proliferative effect. Therefore, the concomitant expression and subsequent co-operative functions of GATA-1 and FOG-1 may play crucial roles in the enhanced proliferation of megakaryocytes. In contrast, impaired terminal differentiation was observed even in the GATA-1-null condition. There are two possible explanations for this effect: firstly, FOG-1 alone plays important roles in this impairment or secondly, a redundant function of GATA-2 compensates for the function of GATA-1. In either case, it is conceivable that the molecular functions of FOG-1 in proliferation and differentiation can be distinguished with respect to the binding partner.
Both the GATA-1-deficient megakaryocytes and FOG-1-expressing wild-type megakaryocytes displayed poor proplatelet formation (Figs 6 and 4, respectively). These data show that GATA-1 is necessary for proplatelet formation and that FOG-1 represses proplatelet formation. It has been reported that FOG-1 activates or inhibits the transcriptional activity of GATA-1 in various gene expression systems (Tsang et al. 1997; Fox et al. 1999; Gaines et al. 2000; Kawabata et al. 2001; Wang et al. 2002). The transcriptional interference of FOG-1 with GATA-1 may account for the reduced proplatelet formation. Taken together, our results show that FOG-1 changes its biological activity according to the differentiation status during megakaryopoiesis, and that different co-operative partners are required for FOG-1 function at different cell stages.
Functional association between FOG-1 and CtBP
CtBP is a broadly expressed corepressor protein that binds to the Pro-X-Asp-Leu-Ser (PXDLS) motifs that are present in many transcription factors, including Kruppel, Net, and the adenoviral E1A (Turner & Crossley 1998; Criqui-Filipe et al. 1999; Postigo & Dean 1999). FOG-1 has been reported to function as a repressor of GATA-1-activated transcription (Fox et al. 1999; Kawabata et al. 2001). This repression is mediated by a small region of FOG-1 (residues 724–834), within which the CtBP-binding motif is located. The binding of FOG-1 to CtBP appears to be essential for transcriptional repression, since FOG-1CtBP does not efficiently repress GATA-1-activated transcription. These molecular data suggest the biological importance of the interaction between FOG-1 and CtBP.
Experiments using FOG-1CtBP have provided some insights into the molecular functions of FOG-1. FOG-1CtBP rescued erythropoiesis and megakaryopoiesis of a FOG-1-null cell line, and showed increased erythropoietic rescue, as compared to the wild-type FOG-1. In addition, mutant mice, into which the FOG-1CtBP mutation had been introduced at the FOG-1 locus, did not show abnormal erythropoiesis (Katz et al. 2002). In Xenopus, erythropoiesis was inhibited by the over-expression of xFOG, mammalian FOG-1, and FOG-2 (Deconinck et al. 2000). Interestingly, the mutated FOG-2, which is unable to bind to CtBP, functioned as a better inducer of erythropoiesis.
We did not observe any enhancement of erythroid differentiation as a result of the over-expression of FOG-1. Instead, FOG-1 impaired significantly the proliferation of erythroid cells at all differentiation stages. This inhibitory effect was independent of the interaction of FOG-1 with CtBP, since the mutated FOG-1CtBP showed similar reductions of erythroid cell proliferation. Although FOG has been reported to function as a repressor of red blood cells in both the mouse and Xenopus, the molecular mechanisms of repression seem to differ in terms of CtBP association. Although GATA-1 expression was reduced by FOG in Xenopus, it was not affected by FOG in our system. These results suggest that the molecular functions of FOG are different in mammals and amphibians. Alternatively, the data from Xenopus may reflect the effect of FOG-1 on the differentiation from mesoderm to haematopoietic cells, but not that of erythroid differentiation.
The inhibition of ES cell proliferation by FOG-1 was independent of GATA factors, since GATA factors 1–6 were not expressed in the ES cells. In addition, this inhibition was mediated by the binding of FOG-1 to CtBP, since FOG-1CtBP had no effect on ES cell proliferation. These data show that FOG-1 functions as a cell proliferation repressor in combination with CtBP, and that FOG-1 function does not require GATA factors. Thus, FOG-1 induces impaired proliferation of haematopoietic cells and ES cells, while the molecular function of FOG-1 varies among the different cell differentiation stages.
Usefulness of ES cell differentiation for scrutinizing transcription factor function
In different cell differentiation contexts, FOG-1 either inhibits or enhances cell proliferation through different molecular mechanisms. This type of differentiation context-dependent function is not amenable to analysis with conventional loss- or gain-of-function experiments. In general, loss of function experiments gives more robust conclusion than over-expression experiments. Since conventional over-expression of genes by retroviral vectors reveals only limited aspects of gene function. First, it is difficult to control the switching on and off of gene expression by the retroviral vector. Thus, it is almost impossible to induce differentiation stage-specific or time-course-selected expression of the genes. Second, only the relatively long-term effects of the genes can be analysed, since the selection of infected cells takes a considerable amount of time. Third, we cannot exclude the possibility that target cells that are infected with retroviral vectors have altered functions and properties.
The experimental system described in this report overcomes these drawbacks. The time-course-dependent and various other functions of FOG-1 are clearly evident in this experimental system. Thus, conventional over-expression experiments are not adequate for elucidating the functions of transcription factors. We are currently analysing the functions of GATA-1, GATA-2, and Runx-1, all of which show cell differentiation context-dependent functions (data not shown). In some cases, conventional loss of function experiments is not sufficient to analyse the differentiation stage specific function of the genes. For example, recent conditional loss-of-function experiments with transcription factors, such as SCL and Runx-1, have shown that these genes are essential for early development of haematopoiesis but dispensable for adult haematopoiesis (Mikkola et al. 2003; Ichikawa et al. 2004). Our experimental system easily turns on and off the genes in a stage specific manner. We believe that conditional and stage restricted over-expression of various genes could provide reasonable results which are difficult to obtain by even conditional gene targeting analysis.
In Xenopus, xFOG repressed erythropoiesis but a study using a mouse erythroid cell line showed that erythroid differentiation (Deconinck et al. 2000). This discrepancy is attributable to species difference or different experimental system. Our present data strongly support the latter possibility. Because our data are well correlated with the data of xFOG but not consistent with that of mouse erythroid cell line (Tsang et al. 1997; Crispino et al. 1999). Thus, the gene functions revealed by studies using cell lines do not necessarily reflect the authentic functions of these genes. In addition, the combination of gene targeting and conditional gene expression constitutes a powerful tool for examining the combinatorial functions of transcriptional activators.
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Experimental procedures |
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The Tet-regulatable FOG-1 or FOG-1CtBP construct was generated by inserting mouse FOG-1 or FOG-1CtBP cDNA into the EcoRI site of pUHD10-3.IRES–EGFP (Era & Witte 2000).
FOG-1CtBP was generated as follows. The SacI-EcoRI fragment of murine FOG-1 cDNA was inserted into the SacI and EcoRI sites of pBluescript (Stratagene, La Jolla, CA, USA). The PIDLSK sequence motif was mutated by PCR to PIASSK (pBS-mutFOG), using the protocol of the Quikchange Site-Directed Mutagenesis Kit (Stratagene, CA, USA). This mutation has been previously described (Schaeper et al. 1995). Representative clones were subjected to automated fluorescent sequencing with the T7 and M13-reverse primers. This mutant fragment was integrated with the remainder of the EcoRI-SacI fragment of FOG-1 cDNA.
Cell cultures and differentiation induction
E14tg2a ES cells and their clones were utilized in this study. These cells were maintained as previously described (Niwa et al. 1998). The culturing of OP9 stroma cells and the in vitro coculturing of ES cells with OP9 cells, to induce differentiation to haematopoietic cells, were performed as previously described (Nakano et al. 1994, 1996). Briefly, ES cells were transferred on to OP9 stromal cells in 6-well plates at a density of 104 cells per well. These induced cells were trypsinized at day 5, and 105 cells were seeded on to fresh OP9 cells. 2 U/mL EPO or 10 ng/mL TPO were added to support the differentiation and proliferation of erythroid cells and megakaryocytes, respectively.
Colony forming unit assays were performed as follows. First, the ES cells were induced into becoming haematopoietic cells by coculture with OP9 cells in a medium that contained 10 ng/mL TPO until day 8. On day 8, the induced cells were transferred into the methylcellulose culture medium Methocult M3232 (Stemcell Technology, Vancouver, BC, Canada) that was supplemented with 10 ng/mL TPO. The colonies were counted seven days after transfer to the methylcellulose medium.
Establishment of Tet-regulated ES cell clones
The tetracycline regulatory system (Gossen & Bujard 1992) was used to obtain ES cell clones that conditionally expressed FOG-1. The expression of the gene was driven by a modified version of the Tet system, as developed by Era & Witte (2000). The production of Tet-regulated clones was carried out essentially as described (Era & Witte 2000). Breifly, the E14tg2a ES cell clone ES2-1, which expresses constitutively the modified Tet-regulated transactivator under the control of the CAG promoter, was established. Then, the linearized plasmids of bicistronic Tet-regulatable FOG-1 or FOG-1CtBP genes (30 µg) were co-transfected with the neomycin plasmid pPGKneo (1 µg) into 8 x 106 ES2-1 cells. For gene introduction, electroporation was performed at 0.23 kV and 500 µF using the Gene Pulser II (Bio-Rad, Hercules, CA, USA). The cells were selected using 200 µg/mL G418 (Sigma, St Louis, MO, USA) for 7–10 days in the presence of 1 µg/mL tetracycline (Sigma). The addition of tetracycline was essential, to avoid the expression of Tet-regulatable genes in ES cells. The clones whose EGFP expression was tightly regulated by tetracycline were selected. Among these clones, the expression of the desired genes was examined by Western blotting, as described below.
Tet-regulated GATA-1 null ES cells were established as followed. Targeting vector was designed as previously described (Fujiwara et al. 1996). The targeting construct was linearized and electroporated into the E14tg2a ES cell clene ES2-1. cDNA of FOG-1 or FOG-1CtBP was introduced into the GATA-1 null ES cells as described above. Two clones were examined.
At least two independent clones, in which FOG-1 or FOG-1CtBP expression was tightly regulated by Tet, were analysed and showed essentially similar results. The data on the representative clones are presented.
Flow cytometry
The cells were harvested by mild pipetting, resuspended in Ca- and Mg-free phosphate-buffered saline (PBS) that contained 5% foetal calf serum, and stained with the PE-conjugated or biotinylated antibodies. The biotinylated anti-mouse GPV (1C2) antibody was purchased from Seikagaku Corporation (Tokyo). The biotinylated anti-TER-119 antibody was a kind gift from Dr T. Kina (Kyoto University, Kyoto, Japan). The biotinylated antibodies were visualized using PE-conjugated streptavidin (PharMingen, San Diego, CA, USA). The PE-conjugated anti-mouse CD71 antibody was purchased from eBioscience (San Diego, CA, USA). For the polyploidization analysis, the cells were suspended in 100 µL PBS that contained 0.1% Triton X-100, 0.2 mg/mL RNase A, and 50 ng/mL propidium iodide. The stained cells were analysed using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). For cell cycle analysis, the cells were suspended and analysed by the same method as for the polyploidization analysis. The cell cycle analysis was carried out using the ModFiTTM LT software (Becton Dickinson).
RT–PCR
Total RNA was recovered using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 100 ng of total RNA was used for cDNA synthesis. Reverse transcription was performed using the ThermoScript RT–PCR system (Gibco BRL, Rockville, MD, USA). The primer sequences and PCR conditions for GATA-1 to GATA-6, FOG-1, and GPDH have been previously described (Suwabe et al. 1998; Tsang et al. 1998).
Western blot analysis
The ES cells or the induced blood cells (2 x 105) were suspended in 15 µL of a solution that contained 0.5% NP40, 100 mM NaCl, 1% glycerol, 1 mM ethylenediamine tetraacetic acid, 1 mM phenylmethylsulphonylfluoride, and 20 mM Tris–HCl (pH 8.0). The protein samples were electrophoresed on a 7.5% SDS–Tris-glycine gel (BIO CRAFT, Tokyo), and transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA, USA). The membranes were probed with the goat polyclonal anti-FOG-1 antibody (M-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-goat IgG antibody (Zymed, San Francisco, CA, USA) was used as the secondary antibody. The membranes were stained with the enhanced chemiluminescence detection kit (Amersham Pharmacia, Buckinghamshire, UK).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: tnakano{at}patho.med.osaka-u.ac.jp
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References |
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Received: 12 June 2004
Accepted: 31 August 2004
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