Human Molecular Genetics, 2002, Vol. 11, No. 8 893-903
© 2002 Oxford University Press
Activation of the ß-like globin genes in transgenic mice is dependent on the presence of the ß-locus control region
1Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA 2Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA 3Organon Pharmaceuticals Inc., Seattle Region, W. Orange, NJ 07052, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The ß-globin locus control region (LCR) is a powerful regulatory element required for high-level globin gene expression. We have generated transgenic mouse lines carrying a ß-globin locus yeast artificial chromosome lacking the LCR to determine if the LCR is required for globin gene activation. ß-Globin gene expression was analyzed by RNase protection, but no detectable levels of
-, 
- and ß-globin gene transcripts were produced at any stage of development. These findings suggest that the presence of the LCR is a minimum requirement for globin gene expression. Next, we tested whether the LCR is necessary to activate globin gene expression in a -globin promoter mutant that causes hereditary persistence of fetal hemoglobin (HPFH). ß-YAC transgenic mice carrying the -117 HPFH mutation and a HS3 core deletion that specifically abolishes -globin gene expression during definitive erythropoiesis were produced to test whether the -117 A promoter is activated in the absence of interaction with the LCR. In four transgenic mouse lines, -globin gene expression was absent in adult erythrocytes, suggesting that an interaction between the -globin gene promoter and the LCR is required for gene activation even when the promoter contains an HPFH mutation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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High-level expression of the ß-globin gene cluster is dependent upon the presence of the locus control region (LCR) (1), a powerful regulatory element physically characterized by a series of five DNase I-hypersensitive sites (HS1–HS5) located 6–22 kb upstream of the ß-globin gene cluster (1–4). The importance of the LCR for normal globin gene expression was revealed by naturally occurring deletions that removed sequences upstream of the ß-like globin genes; invariably, these deletions result in changes in the chromatin structure and in transcriptional silencing of the ß-globin locus (5–8). Early studies have shown that transgenic mice carrying human globin transgenes lacking the LCR either fail to express the globin transgene or express them inconsistently at low levels, indicating strong negative influences of the surrounding chromatin at the site of transgene (9–11). In contrast, addition of the LCR or of individual HSs to the transgenic constructs resulted in copy-number-dependent expression, independent of the site of transgene integration in the mouse genome (1, 12–14). Thus, the LCR is functionally defined in transgenic mice as a regulatory element capable of providing high-level, copy-number-dependent expression upon a linked gene independent of the site of integration.
Examination of the LCR in transgenic mice has shown that the majority of the LCR activity is associated with HSs, and, more specifically, to the core elements of the HSs; these core elements range in size between 200 and 400 bp (15–18; reviewed in 19). Individual HSs display developmental specificity (20,21). A considerable amount of information has been obtained about the function of the HSs by characterizing mutations of the HSs introduced in the context of the human or mouse ß-globin locus. Deletion of large fragments (flanking sequences plus core element) of either HS2 or HS3 from the endogenous murine ß-locus or a human ß-globin locus yeast artificial chromosome (YAC) resulted in only modest reduction in globin gene expression in mice (22–24). These results suggested that the LCR contains functionally redundant elements, so that the function of the deleted HS is provided by the remaining HSs. In contrast, deletion of only the HS core elements resulted in severe reduction of globin gene expression (21,25–27). Deletion of the core element of HS3 in the context of a 248 kb ß-YAC resulted in a very characteristic phenotype consisting of total absence of -globin gene expression but normal -globin gene expression in the embryonic yolk sac and total absence of -globin gene expression in fetal liver erythropoiesis (21). These results indicated that the HS3 core element is required for activating the -globin gene during embryonic erythropoiesis and the -globin genes during definitive erythropoiesis in the fetal liver.
In the present study, we analyzed transgenic mice carrying a human ß-globin YAC, from which the LCR has been deleted to determine if it is dispensable for the activation of the ß-globin genes. We wished also to know if the LCR is required when a downstream -globin gene carries a mutation that increases -globin promoter strength resulting in increased -globin gene expression in the adult. The most characteristic examples of such mutations are the variants of hereditary persistence of fetal hemoglobin (HPFH) in which -globin gene expression in the adult is increased 20–50-fold above baseline levels. One of these mutations is a single-point base-pair substitution of two nucleotides upstream of the duplicated CCAAT box region at position -117 of the A-globin gene promoter that results in the production of 10–20% HbF in adult life (28,29). Transgenic mice carrying this HPFH mutation have high levels of postnatal -globin gene expression and therefore present the phenotype of HPFH (30–32). Since the deletion of the HS3 core element is associated with the complete absence of -globin gene expression in adult mice, we deleted the HS3 core from a ß-locus YAC containing the -117 A HPFH mutation and used the double 
HS3c/-117 A ß YAC mutant for production of transgenic mice. We found that -globin gene expression was totally absent in the adult, suggesting that an interaction between the promoter and the LCR is required for -globin gene activation even when the promoter contains a mutation that increases promoter strength.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Analysis of human globin gene expression in
LCR transgenic miceWe have previously shown that the 248 kb ß-YAC transgene is frequently deleted at the 5' or 3' end, most likely the result of extensive in vitro manipulation of this large DNA fragment prior to microinjection of the mouse oocytes (21,27,33). In spite of these rearrangements, when the LCR and ß-locus genes remain intact, they are properly regulated during development and have normal levels of expression (33). Because of the potential for structural rearrangements, extensive structural analysis of the YACs integrated into the mouse genome has become a prerequisite to functional analysis of YAC transgenics.
During the production of ß-YAC transgenic lines, three ß-YAC transgenic mouse founders had the entire LCR deleted but most of the ß-globin gene cluster intact; these founders were subsequently bred to establish transgenic lines (Fig. 1). The continuity of each of the integrated ß-YAC fragments was determined by a detailed structural analysis as described in Materials and Methods. Line LCR-1 has two copies, a 230 kb SfiI copy containing sequences from A to 3'HS1 (identified by probe DF10) and a 150 kb fragment containing only the ß-globin gene through to the HPFH6 breakpoint. Line LCR-2 has a single 170 kb SfiI fragment containing sequences from A to the HPFH6 breakpoint. Line LCR-3 has two SfiI fragments, of 160 and 180 kb in size, that contain sequences from the -globin gene to the HPFH3 breakpoint. Studies of human globin gene expression during development included developmental time points corresponding to embryonic (day 9 postconception), fetal (days 12 and 16 postconception) and adult (
3 weeks after birth) globin gene expression. Total RNA was isolated from yolk sac, liver and blood samples of individual animals and subjected to RNase protection analysis. We analyzed at least three animals per time point and per line to minimize experimental error and to assess the variation in globin gene expression between littermates. The resulting signals were quantitated by PhosphorImager analysis, and expression levels were calculated as a percentage of murine 
- plus 
-globin mRNA per copy. Human globin gene expression can be accurately measured as low as 0.5% of murine - plus -globin mRNA (per transgene copy) by RNase protection analysis.
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We were unable to detect any human globin transcripts at any time during development in any of the three
LCR transgenic mouse lines. Also, we were unable to detect human globin protein by staining of placental blood smears from day 9 and 12 embryos, and fetal liver preparations from day 12 and 16 fetuses with anti-, - and ß-globin-chain fluorescent monoclonal antibodies (data not shown). We conclude that in the absence of the LCR, the human globin genes are transcriptionally silent throughout development in transgenic mice.
Production of HS3c/-117 Am ß-globin locus YAC transgenics
To test whether the LCR is required for the activation of an HPFH gene, we produced a double mutant ß-YAC that links the HS3 core deletion to the single base G-to-A substitution at position -117 in the distal CCAAT box of the A-globin promoter in the context of the 248 kb ß-YAC (Greek HPFH mutation, Fig. 2). The HS3c/-117 Am ß-YAC was produced by homologous recombination of the HS3 core deletion following transformation of a yeast strain containing the -117 Am ß-YAC (Fig. 2) (31).
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The four transgenic lines that we produced were analyzed for the HS3 core deletion and for the presence of the -117 base substitution in the A
m-gene promoter. By Southern blot hybridization analysis, we were able to confirm the HS3 core deletion and also the presence of HS4, which resides upstream of the 5'SfiI site used in our structural analysis. The deletion of the HS3 core was accomplished by site-directed mutagenesis that created EcoRI sites bracketing the 234 bp core element of HS3; subsequent digestion with EcoRI and ligation resulted in the loss of the core element while leaving a diagnostic EcoRI site (21). HS2–HS4 reside on a 10.4 kb EcoRI fragment in the wild-type locus. When the HS3 core deletion is present, digestion with EcoRI yields two fragments of 4.5 and 5.6 kb in size. HS4 resides on the 4.5 kb fragment, confirming that each line (with the exception of the HS3 core deletion) contains an otherwise-intact LCR (Fig. 3A).
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The G-to-A base substitution at position -117 of the distal CCAAT box of the A
promoter creates a MseI restriction site that distinguishes the -117 Am promoter from the wild-type promoter. We designed oligonucleotides specific for PCR amplification of the A promoter. Digestion of the 917 bp wild-type PCR product with MseI generates a 390 bp, two 179 bp and a 167 bp fragments. The mutant -117 Am PCR products results in the same fragments, with the exception of the 390 bp fragment, which is restricted into two fragments of 273 and 111 bp. The 273 bp fragment contains a 6 bp deletion in the 5' untranslated region of the A gene that can be used to distinguish Am transcripts from G and wild-type A transcripts. As shown in Figure 3B, all four transgenic lines had the 273 and 111 bp fragments, confirming the presence of the -117 mutation in these lines. These fragments were sequenced to verify the presence of the -117 HPFH mutation and to ensure that no other mutations were introduced (data not shown).
Structural analysis of HS3c/-117 Am ß-globin locus YAC transgenics
We established four double mutant transgenic lines harboring at least one intact ß-YAC copy. Figure 4 shows the results of our structural analysis and graphically illustrates the structure of each of the integrated ß-YAC copies.
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Line AA has four copies: a single intact 140 kb SfiI copy and three additional copies identified only with the HPFH6 probe. The single intact ß-YAC fragment contains a ß-globin locus spanning HS3 through the HPFH6 breakpoint sequence. Copy number analysis determined that there were two copies of this YAC.
Line AB has three SfiI fragments containing human ß-locus sequences, a 135 kb SfiI fragment containing the entire ß-globin locus, and two additional SfiI fragments of 90 and 130 kb in size. The 135 kb SfiI fragment has an intact ß-globin locus that contains the ß-globin gene, includes 3'HS1, and is the lone YAC fragment with LCR sequences linked to the ß-like globin genes. The 90 kb fragment contains HS3, HS2 and possibly sequences to the HPFH6 breakpoint, but an internal deletion has removed all of the globin genes. The 130 kb fragment contains the A gene through the ß-globin gene, but is not expected to contribute to globin gene expression owing to the deletion of the LCR. Thus, in line AB, all globin transcription is expected to originate from only the 135 kb fragment.
Line AC has SfiI fragments ranging from 100 to 280 kb in size. There are at least three intact ß-globin loci: 140 and 160 kb fragments that contain sequences from HS3 of the LCR to the HPFH6 breakpoint and a 125 kb fragment with a deletion of sequences downstream of the ß-globin gene. A 100 kb fragment has the LCR linked to the -globin gene, but is deleted of the remaining globin genes. This YAC copy is expected to contribute to -globin gene expression during embryonic erythropoiesis. A 130 kb fragment has the LCR linked to the - and -globin genes. The -, G- and A- genes are expected to be expressed. A 280 kb fragment that links the LCR to globin sequences through the 
ß psuedogene would be expected to express both the - and the two -globin genes. Four fragments (shown by lines on the right of the diagram) contain only sequences 3' of the ß-globin gene. Thus, this transgenic mouse line has the potential to express globin chains from at least six copies of the -globin genes, five copies of the G- and A-globin genes, and three copies of the 
- and ß-globin genes.
Line AD has four distinct SfiI fragments, all of which contain an intact LCR, but have varying deletions of the 3' end of the ß-YAC. The two intact ß-globin loci are of 130 and 160 kb in size. The 160 kb fragment contains sequences up to, but not including, the HPFH6 breakpoint. The 130 kb fragment is deleted of sequences 3' to the ß-globin gene. The 115 kb fragment has the LCR linked to the -globin gene, but deleted of the remaining genes. The 180 kb fragment appears to have an internal deletion that includes the A-globin gene and the ß-pseudogene region, thus juxtapositioning the - and -globin genes next to one another. Two faint signals appear in the and ß lanes, but the difference in intensity of the signals relative to other probe signals leads us to conclude that these signals are a result of cross-hybridization of these globin probes and do not represent actual globin sequences present in this YAC fragment. Thus, line AD has the potential to synthesize globin chains from four -globin genes, two G- and A-globin genes, and three ß-globin genes during development.
Analysis of -globin gene expression in -117 A HPFH mice lacking the core element of HS3
To analyze the expression of individual globin genes in the transgenic lines, we performed RNase protection assays on total RNA to measure levels of human -, - and ß-globin mRNAs, as well as those of the endogenous mouse - and -globin mRNAs that served as internal controls. Total RNA was isolated from yolk sac and blood of multiple day-10 and day-12 postconception F2 embryos from the same litter to minimize experimental error and to control for sample variation. As shown in Figure 5, and summarized in Table 1, -globin expression is undetectable in day-10 yolk sac and day-12 blood in three of four HS3c/-117 Am ß-YAC lines, in agreement with previously published results (21). Also in agreement with previous results (21), -gene expression in the same samples is normal (Fig. 5, Table 1). The average expression of the -globin genes among the four HS3c/-117 Am ß-YAC transgenic lines in day-10 yolk sac and day-12 blood, 19.8%±4.4% and 25.7%±5.8%, respectively, is indistinguishable from that of the control wild-type ß-YAC mice. Thus, the -117 A HPFH mutation had no affect on -globin gene expression during embryonic erythropoiesis.
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We previously showed that the HS3 core deletion resulted in the complete absence of
-globin gene expression during definitive erythropoiesis (21). In contrast, transgenic mice carrying the wild-type ß-YAC express -globin mRNA in the definitive erythroid cells of the fetal liver. The mean -globin mRNA in day-12 and day-14 livers of mice carrying the wild-type ß-globin locus was 20.7%±5.1% and 5.7%±2.4%, respectively, and -globin gene expression continued to decline during fetal life and ultimately switched off after birth (Table 1). No -globin mRNA was detected by RNase protection analysis after postnatal day 7 (33,34). The introduction of the Greek -117 HPFH mutation into the ß-YAC resulted in transgenic mice whose -globin gene expression remained elevated during fetal life and delayed the - to ß-globin switch. -Globin gene expression continued into adult life, finally averaging approximately 8% of total human globin mRNA as measured by RNase protection analysis (31) and approximately 12% of total human globin protein as measured by high-performance liquid chromatography (HPLC) (32).
Human -globin expression in the yolk sac of the HS3c/-117 Am ß-YAC mice was the same as in the control mice, supporting our previous evidence that the core element of HS3 is not required for -globin gene expression during embryonic erythropoiesis. -Globin gene expression was present in the day-12 fetal liver samples; however, the overall level was strikingly reduced compared to normal controls (Fig. 5, Table 1). Staining of fetal liver preparations with anti- fluorescent antibodies indicated that the bulk of this expression derived from the embryonic erythroblasts that contaminate the fetal liver (Fig. 6A and B). However, the fluorescent anti- labeling also showed several definitive erythroblasts (identified in the figure by arrows) expressing -globin (Fig. 6A and 6B). These results suggest that the presence of the -117 A mutation allow a minor degree of -globin gene expression in the fetal liver in the HSc/-117 A ß-YAC mice. This expression appears to be stochastic, since only a few erythrocytes were stained with the anti--globin chain antibody.
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-Globin mRNA was not detectable by RNase protection analysis (Fig. 5, Table 1) and -globin protein chains were not detectable by staining blood smear preparations with anti--globin chain fluorescent monoclonal antibody in the red cells of adult HSc/-117 A ß-YAC transgenic mice (not shown).
ß-Globin gene expression in HS3c/-117 ß-YAC transgenic mice is decreased and position dependent
In agreement with our previous findings in the HS3c lines (21), the levels of ß-globin gene expression in HS3c/-117 ß-YAC transgenic mice were significantly lower than in wild-type control mice and were strongly influenced by the position of integration of the transgene. ß-Globin expression ranged from 10.7%±0.8% to 35.9%±11.6% and was considerably less than the wild-type control of 101.6%±16.7% in adult blood (Table 1). In wild-type ß-YAC transgenic mice, there is a small variation in the ß-globin gene expression levels, indicating that the ß-globin genes are protected from the negative effects of the surrounding chromatin. A statistical measure of the impact of the surrounding chromatin on globin gene expression is the coefficient of variation (CV), a value expressed as the standard deviation in per-copy expression as a fraction of the mean (
/µ), with values smaller than 0.5 denoting statistically insignificant degrees of variation in globin gene expression between transgenic mouse lines. Coefficients of variation for per-copy ß-globin gene expression in day-12 and day-14 fetuses and adult mice were 0.46, 0.61 and 0.72, respectively, suggesting that the LCR, in the absence of the HS3 core element, is unable to provide for position-independent ß-globin gene expression in day-14 fetuses and adults.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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When YACs are used for production of transgenic mouse lines, rearrangements of the transgenes occur most likely during in vitro manipulation of the transgene or during the process of transgene integration into the murine genome. Such rearranged YACs can create many problems in the interpretation of the data in the absence of a very detailed analysis of the integrated transgenes. For example, YAC fragments integrated in different chromosomal sites can provide the erroneous information of an intact locus if conventional Southern hybridization analysis or polymerase chain reaction (PCR) of the integrated DNA is done. For this reason, we have developed a methodology that allows us to examine the structural integrity of the integrated YACs and decide with great confidence whether the integrated copies are intact or whether they have suffered deletions or rearrangements (33,35). Most of the ß-globin locus resides on a 140 kb SfiI restriction enzyme fragment contained within the 248 kb ß-locus YAC. As shown in Results, fractionation by pulsed-field gel electrophoresis (PFGE) of the high-molecular-weight DNA digested with SfiI followed by Southern blot hybridization of multiple PFGE lanes with probes spanning the entire ß-globin locus can identify, in a linear fashion, all the globin-gene-containing SfiI fragments that are integrated into the genome of the transgenic mice. This assay allows us to determine to what extent the ß-globin locus of each SfiI fragment is intact and thus accurately correlate the structure of the locus to globin gene expression.
Three transgenic lines carrying the human ß-globin genes deleted of the LCR (LCR ß-YAC) failed to express the human -, - and ß-globin genes during any stage of development. These results are similar to those obtained from the analysis of the human thalassemia mutants due to LCR deletions (reviewed in 19). The evidence for lack of gene expression in the LCR ß-YAC transgenic lines is based on RNase protection analysis of total RNA, an assay that in our laboratory can detect human transcripts at levels of 0.5% of endogenous murine globin mRNA (corrected for transgene copy number) (21,36). A previous study of transgenic mice carrying 4–15 copies of a 40 kb human ß-globin gene cosmid containing the G-, A-, - and ß-globin genes reported that these genes were expressed and developmentally regulated in the absence of the LCR (37). However, the RNase protection assays in that study utilized from 10- to 125-fold more RNA in their reactions compared with the present study and detected levels of human globin mRNA that did not exceed 1% of endogenous murine globin mRNA (37). The significance of such rare globin transcripts remains unclear.
Recent studies in mice carrying a deletion of the endogenous murine ß-locus LCR suggested that the LCR is necessary for normal levels of globin gene expression, but it is not required for chromatin opening (38–40). The deletion of HS1–HS6 of the LCR resulted in the very low-level globin gene expression (about 1% of endogenous ß-globin), but the ß-globin locus established and maintained a DNase I-sensitive chromatin conformation (39). The difference between the phenotype of the murine ß LCR deletion in which the locus is in the DNAse I-sensitive conformation and the phenotype of the deleted LCR thalassemia mutants is whether the ß-locus chromatin is DNAse I-insensitive remains to be explained. Among the possible reasons for these differences is that in the thalassemia mutants, the deletions are more extensive and perhaps remove upstream insulator elements that normally prevent the spread of heterochromatinization into the human ß-globin locus; alternatively, elements that contribute to ‘opening’ the murine ß-globin locus domain are present throughout the murine ß-locus (39). In another study, deletion of the endogenous murine ß-locus LCR resulted in total absence of ß-globin gene expression in about 80% of the erythrocytes of the LCR-/- mice but presence of ß-gene expression ranging from very low to almost normal in the remaining red cells (T. Townes personal communication). The differences in the phenotype of the LCR-/- mice between these two studies remain unclear. Staining of the peripheral blood of our transgenic lines carrying the human LCR deletion with anti-human ß-chain monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) failed to detect red cells expressing human ß-globin.
Our studies indicate that the presence of the LCR is also necessary for globin gene expression in the non-deletional mutants of HPFH. Typically these non-deletional forms of HPFH are due to mutations of the G- or A-globin gene promoters, and they are characterized by elevation of HbF in adult life without any hematological abnormalities. In our study, we used the -117 A G-to-A mutant of HPFH because the phenotype of this HPFH has been reproduced in transgenic mice carrying either a cosmid (30) or a ß-locus YAC (31,32). The phenotype of HPFH mutants has been attributed either to an increase of the strength of the -gene promoter that allows the mutant promoter to interact with the ‘adult-type’ transcriptional environment of the erythroid cell or to inhibition, by the mutation, of binding of adult-stage-specific suppressors (which normally bring about -gene silencing) (41–43). Studies in transgenic mice suggest that an increase in promoter strength is the most likely mechanism of -gene expression in adults carrying the -117 A HPFH (36). It is likely that the non-deletional HPFH mutations exert their effects by increasing the probability that the -gene promoter will interact with the LCR in the adult life, but direct evidence for this is lacking.
Previous investigations have shown that there is developmental specificity in the interaction between the HSs of the LCR and the globin promoters. HS3 is required for gene expression in embryonic cells and for -gene expression in the cells of adult erythropoiesis (21), while HS4 is required for ß-globin gene expression in definitive erythroid cells (20,21) and, in the presence of HS3, contributes to the level of gene expression in the fetal erythroid cells (21). These previous results predict that if indeed the -117 A HPFH promoter increases the probability of interaction with the LCR in the adult erythroid cell environment, this interaction will be mediated through HS4. In contrast, we found that the double HS3/-117 A ß-YAC transgenic mutants display only minimal -gene expression in fetal liver erythropoiesis and no -gene expression in adult erythropoiesis. These results indicate that, similarly to the wild-type -gene promoter, the -117 A HPFH promoter interacts with the HS3 of the LCR in adult erythropoiesis. Further studies of the interaction between the LCR and the -gene promoter of the -117 A HPFH ß-YAC transgenic mice may identify the specific elements of the HS3 that participate in the LCR/-promoter interactions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the
HS3c/-117 ß-YAC transgenePlasmid pRS
HS3c(1.6), a yeast-integrating-plasmid (YIP) containing the 1.6 kb HindIII fragment deleted of the HS3 core element (21), was linearized with the restriction enzyme SpeI and transformed into spheroplasted Saccharomyces cerevisiae strain AB1380 containing the -117 Am ß-YAC (31). Transformants were selected for uracil prototrophy on complete medium, and correct recombination was determined by Southern blot analysis. Spontaneous excision of the YIP was induced by overnight growth in non-selective rich medium (yeast–peptone–dextrose). The yeast cells were plated on 5-fluoroorotic acid plates to select for loss of the URA3 gene residing on the YIP vector, resulting in 5-fluoroorotic acid resistance. Deletion of the HS3 core element was confirmed by Southern blot analysis.
YAC purification and production of transgenic mice
The yeast strain containing the HS3c/-117 ß-YAC was grown in 200 ml of YPD (1% yeast extract, 2% peptone, 2% dextrose) overnight. The saturated culture should be approximately 1 x 108 cells per ml. The yeast were embedded in agarose and lysed as previously described (44). Preparative plugs were loaded on a 0.5% Seakem Gold agarose gel (BioWhittaker Molecular Applications, Rockland, ME), and the DNA was fractionated by PFGE on CHEF DRII apparatus (Bio-Rad, Hercules, CA) in 0.5x TBE (44.5 m
Tris, 44.5 m boric acid, 1 m ethylene diamin tetraacetic acid, EDTA) at 200 V with a 60 s switch for 17 hours at 12°C. The first two lanes containing yeast chromosomal marker (New England BioLabs, Beverly, MA) and yeast preparative plugs were stained with ethidium bromide to visualize the HS3c/-117 ß-YAC and determine the migration distance. A slice containing the YAC DNA was rotated 90° relative to the original electrophoresis direction and electrophoresed in a 4% low-melting-point agarose gel (NuSieve GTG; BioWhittaker Molecular Applications, Rockland, ME) in 0.5x TBE at 47 V for 15 hours to concentrate the DNA. The YAC DNA runs into approximately 8 mm, and the DNA was sliced out and equilibrated in a 100x volume of a high-salt buffer (10 m Tris–HCl (pH 7.5)–250 µ EDTA–100 m NaCl) for 1 hour at room temperature without agitation. The gel slice was blotted on Whatman #1 filter paper (Whatman Inc., Clifton, NJ) to remove excess solution, placed in a microfuge tube, and placed at 68°C for 10 min to melt agarose. The tube was immediately placed at 42.5°C for 5 min, followed by the addition of two units of ß-agarose (New England BioLabs, Beverly, MA) per 100 mg of agarose, and the digestion was allowed to go overnight. The integrity of the isolated YAC was checked by PFGE and the concentration was determined by fluorometry (Pharamcia, Piscataway, NJ). The YAC DNA was diluted to a final concentration of 2.0 ng/µl with high-salt buffer and filtered through a 0.22 µm pore Acrodisk (Gelman, Ann Arbor, MI) just prior to injection into mouse oocytes. The purified and filtered YAC was injected into fertilized mouse eggs (B6/C3F1) and then transferred to pseudopregnant foster mothers (B6/D2F1). Founder animals were identified by Southern hybridization slot blot of DNA isolated by tail biopsies and probed for the ß-globin gene. The transgenic founders were bred to produce F1 progeny, and the F1 progeny were bred with non-transgenic mice (B6/D2F1) for staged pregnancies that were interrupted at postconception days 10, 12 and 14 and to produce F2 progeny.
Determination of transgene copy number
Agarose plugs containing high-molecular-weight DNA from transgenic mouse livers were digested overnight with restriction enzymes and fractionated by agarose gel electrophoresis, and blotted onto zeta-probe positive-charged nylon membrane (Bio-Rad, Hercules, CA). Transgene copy number was determined by comparing HS2, -, A- and ß-globin gene hybridization signals with the endogenous murine Thy1.1 signals by Southern blot hybridization analyses as previously described (27). The radioactive signals were measured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the ratio of human globin sequences to Thy1.1 (corrected for a haploid genome) were calculated to determine transgene copy number.
Structural analysis of LCR ß-YAC and HS3c/-117 Am ß-YAC transgenic mice
High-molecular-weight DNA embedded in agarose was prepared as previously described (27). Twelve 4 mm slices of each transgenic mouse line and an agarose slice containing DNA from a MEL cell line containing a single intact ß-YAC were digested with SfiI at final concentration of 100 units/ml overnight at 50°C in a total volume of 200 µl. The digested DNAs were fractionated by PFGE on a 1% (w/v) SeaKem Gold GTG agarose gel (BioWhittaker Molecular Applications, Rockland, ME) in 0.5x TBE, 200 V, 14 s switch, for 22 hours and at 14°C. The DNA was capillary-transferred overnight onto zeta-probe positive-charged nylon membrane (Bio-Rad, Hercules, CA) with 10x SSC. The nylon membranes were cut into strips representing individual lanes and each strip was hybridized with a different radioactive probe spanning the ß-globin locus from HS3 to the HPFH6 breakpoint. After hybridization, the strips were reassembled and subjected to autoradiography. The radiolabeled fragments used as probes for the structural analyses are as follows: 0.7 kb PstI HS3, 1.9 kb HindIII HS2, 1.8 kb XbaI HS1, 3.7 kb EcoRI -globin gene, 2.4 kb EcoRI fragment 3' of the A-globin gene, 1.0 kb EcoRV ß region, 2.1 kb PstI fragment upstream of the -globin gene, 0.9 kb EcoRI-BamHI fragment 3' of the ß-globin gene, 1.4 kb XbaI DF10 (3'HS1), 1.9 kb BglII HPFH3, 0.5 kb HindIII H500, and 1.5 kb EcoRI-BglII HPFH6. The fragments were radiolabeled using a Decaprime II random probe labeling kit using the manufacturer's instructions (Ambion, Austin, TX).
Measurement of globin mRNA synthesis
Total RNA was isolated from F2 transgenic embryos, fetuses and adults using the Total RNA Isolation system following the manufacturer's instructions (Promega, Madison, WI). Human and murine globin mRNAs were detected by RNase protection analysis and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Template DNAs used to prepare riboprobes to measure -, - and ß-globin mRNA were pT7Hu(188), pT7Am (170) and pT7ßm, repectively (45). To distinguish between G- and A-globin mRNA, the DNA template was generated from plasmid pT7Aexon1wt. The source and amount (in parentheses) of isolated RNAs used in RNase protection assays are as follows: day-10 yolk sac (1000 ng), day-12 liver (500 ng), day-12 blood (80 ng), day-14 liver (500 ng), and adult blood (50 ng).
Immunofluorescent detection of human globin chains
Day-12 and day-14 fetal liver cell preparations were fixed and stained with -, - or ß-globin-specific monoclonal antibodies. A second antibody, goat F(ab')2 anti-mouse FITC-conjugated immunoglobulin G (Dupont, Wilmington, DL), that is reactive to the mouse monoclonal antibodies was added to visualize human globin proteins.
Polymerase chain reactions
The A-globin promoter region was amplified using the following A-specific primers: 5'A SP.1 (proximal) 5' CATACCTGAATATGGAATC 3' and 3'A SP.2 (distal) 5' CTGGTCACCAGAGCCTAC 3'. 100 ng of genomic DNA was PCR-amplified using Taq DNA polymerase in Storage Buffer B following the manufacturer's instructions (Promega, Madison, WI). The PCR conditions were as follows: 1 min at 95°C, 1 min at 53°C and 1.5 min at 72°C for 35 cycles.
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ACKNOWLEDGEMENT |
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This work was supported by the US National Institutes of Health grants DK45365 and HL20899.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Tel: +1 206 543 3526; Fax: +1 206 543 3050; Email: gstam@u.washington.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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