Abstract
After screening a bacterial artificial chromosome of human genomic DNA library with human HS-40, ζ-, α-, and θ-globin probes, a 110-kb clone bearing the whole human α-globin gene cluster was obtained and rare restriction endonuclease mapping was performed. The bacterial artificial chromosome DNA was isolated, and transgenic mice were generated. Three founders were detected from 35 newborn mice. The copy numbers were 1, 2, and 2, and the expression of human α-globin genes in various tissues at different developmental stages in the transgenic mice was assayed. The human α-globin mRNA can be detected in bone marrow, kidney, liver, brain, but not in muscle, testis, or thymus. The human ζ-globin genes were switched off, and the α-globin genes were switched at day 11.5 in mouse embryo, indicating that developmental stage-specific expression of the α-like globin genes was properly regulated. The human α-globin mRNA ranged between 17–68% of the endogenous mouse α-globin, suggesting that the expression of human α-globin genes is integration site-dependent in transgenic mice. The ratio of human α2- and α1-globin gene expression in adult transgenic mouse is about 2.5:1 similar to the expression in human.
The human hemoglobin is made up of tetramers, two α-like and two β-like globin chains. In the adult and fetal stages, α-chains combined with β-(HbA, α2β2), δ-(HbA2, α2δ2), or γ-chains (HbF, α2γ2), whereas in the embryo stage, embryonic α-like chains named ζ-chains combined with γ- (Hb Potland, ζ2γ2) or ɛ-chains (HbGower1, ζ2ɛ2). Embryonic ζ-globin is confined to the yolk sac stage of development and thereafter is replaced by two fetal/adult α-globin chains (α2- and α1-globin) (1). The human α-like globin gene cluster spans about 80 kb at the short arm of chromosome 16 and includes seven tandemly linked genes organized in the order of their developmental expression: 5′-ζ-ψζ-ψα2-ψα1-α2-α1-θ-3′ (2). HS-40 is the major control element of α-globin gene family located 40 kb upstream of ζ-globin gene. There are various erythroid-specific and ubiquitous DNA-binding protein binding sites within the 300-bp core region (3, 4), similar to the locus control region of the human β-globin gene cluster (5). Their functions, however, are somewhat different from each other, because HS-40 is designated as a “positive control element” of the human α-globin gene cluster (6).
The developmental switching and tissue-specific expression pattern of α-like globin genes provide a model system for the study of gene regulation. The switching from a single embryonic ζ-globin to two coexpressed fetal/adult α2- and α1- globin takes place at 6–7 weeks of gestation, and two α-globin genes are fully active and retain this level after birth. Understanding α-globin gene regulation may provide an approach to the therapy of α-thalassemia. Transgenic animals and transfected erythroleukemia cells are two useful experimental systems for analysis of the regulation of human α-globin genes (7, 8). In mouse erythroleukemia (MEL) cells containing human chromosome 16, high levels of human α-globin gene expression are observed after chemical induction. Nevertheless, it can express only the adult human α-globin and no developmental switching can be observed in the MEL cells.
Studies in transgenic mice have provided deep insights into the regulation of α-globin expression, but results from single α- or ζ-globin transgenic mice could not reflect the real chromosome environment of the human α-globin gene cluster. Gourdon et al. (9) ligated two cosmids through an oligonucleotide linker to produce a single fragment spanning 70 kb of the human α-globin cluster; the ζ- and α-globin genes were expressed in transgenic embryos. However, such a cosmid may lack some cis-elements beyond HS-40 and the expressed genes. To study the organization and developmental switching pattern of human α-like globin genes, it is necessary to establish transgenic mice containing the whole human α-globin gene cluster.
Yeast artificial chromosome (YAC) has been widely applied in large-fragment transgenic research of the human β-globin gene cluster, but as a vector system (10), YAC is unstable and difficult to be purified, restricting its application in human α-globin gene cluster research.
A bacterial artificial chromosome (BAC) library provides a better alternative for this problem. The average insert of BAC vector is about 150 kb (11), which can cover the whole human α-globin gene cluster. The BAC DNA is a single-copy circular DNA molecule in bacterial cell and is stable and easier to be purified than cosmid and yeast artificial chromosome. The generation of BAC-mediated transgenic mice of whole human α-globin gene cluster may provide more information for understanding the organization and switching pattern of the gene cluster.
In this study, we screened the human BAC library and generated transgenic mice bearing the selected BAC clone containing whole human α-globin gene cluster. Tissue- and developmental stage-specific expression of human α-like globins was observed in the BAC-mediated transgenic mice. The expression level of the human α-globin gene was found to be integration site-dependent in different transgenic lines.
Materials and Methods
Human BAC Library Screening.
The high-density hybrid membranes for the BAC library (Research Genetics, Huntsville, AL) were screened by [α-32P]dCTP-labeled HS-40 and θ-globin probes (gifts from James Shen, Academia Sinica, Taipei, Taiwan). Positive clones were picked from the BAC library according to the hybrid dots on x-ray films. Then mini- and large-scale preparations of BAC DNA were carried out by standard procedures. Selected clones were determined further by Southern blot and restriction endonucleases analyses.
Clone Determination.
The BAC DNA was purified by alkaline lysis and digested with different restriction endocleases including NotI, NruI, PacI, SalI, and RsrII. The digested BAC DNA was separated by pulse field gel electrophoresis (PFGE) on 1% agarose (Roche Molecular Biochemicals), using the 10–250-kb autoprogram of chef mapper (Bio-Rad). Usually, NotI digestion of BAC DNA was transferred to nylon membrane for Southern blot analysis. A physical map of the BAC clone was drawn according to the PFGE and Southern blot results.
Fluorescence in Situ Hybridization (FISH) Analysis of BAC Clone.
About 300 ng of purified BAC DNA was labeled with Biotin-16-dUTP by nick-translation according to the manufacturer's suggestion (1-745-824, Roche Molecular Biochemicals). The labeled probe was hybridized with colchicine-treated human peripheral blood lymphocytes. Hybridization signals were detected with FITC-conjugated avidin followed by two rounds of amplification with biotinylated anti-avidin. Hybridization signals were scored under an Olympus (New Hyde Park, NY) BX-60 fluorescence microscope, and the images were captured on a Photometrics (Tucson, AZ) charge-coupled device (CCD) camera by using applied imaging software.
DNA Purification for Microinjection.
BAC DNA was purified by routine alkaline lysis and CsCl gradient ultracentrifugation (75,000 rpm, 20°C, 4 h) from 1,000 ml of bacterial culture and dissolved in 200 μl of TE buffer (10 mM Tris/1 mM EDTA, pH 8.0). Then 50 μg of BAC DNA was digested by 200 units NotI in a 500 μl volume at 37°C for 10–15 h.
The digested BAC DNA was added to a 0.5 × 5 preequilibrized Sepharose CL-4B column. The column was washed by injection buffer (10 mM Tris⋅HCl, pH 7.5/0.1 mM EDTA, pH 8.0/100 mM NaCl), the washed fractions were collected with a 24-well plate (0.5 ml for each well). Of each fraction, 20 μl was run on pulse field gel electrophoresis to identify the appropriate fractions. The fractions containing the linearized BAC DNA were collected and used for microinjection.
Generation of Transgenic Mice.
The murine zygotes were from C57BL/6J-mated KM female mice; the fosters were KM female mice. The purified BAC DNA was diluted to 1 ng/μl for pronuclear microinjection into murine zygotes. The injected zygotes were cultured in M16 culture medium for 4–5 h, and healthy zygotes were selected for oviduct transplantation.
Detection of Founder Mice.
Mouse tail genomic DNA was purified by phenol/chloroform until the mice were 4–6-weeks-old. First, the transgenic founders were rapidly screened by PCR, using 5′-TTCGTCATAATATGGGTTTTT-3′ and 5′-TGTGGGGAAAGAAATTAAATTA-3′ as primers. The PCR parameters were 94°C for 40 s, 58°C for 40 s, 72°C for 40 s (30 cycles). The size of amplified fragment was about 500 bp, containing the HS-40 sequence. Then the founder mice were identified further by Southern blot analysis by using human HS-40, ζ-, α-, and θ-globin genes as probes. (The HS-40 and θ-globin probes were gifts from J. Shen; ζ- and α-globin probes were cloned by the National Laboratory of Medical Molecular Biology). Positive founders were bred to KM/ICR mice, and copy nos. of the founder mice were quantified by Southern blot, using a PhosphorImager (Molecular Dynamics).
Total RNA Isolation and RNase Protection Assay.
Yolk sacs and fetal livers from different developmental stages of transgenic mice embryos were collected. After determination by PCR, total RNA was extracted from the yolk sac, fetal liver, bone marrow, and other tissues of adult mice by Trizol reagent (GIBCO/BRL) and dissolved in diethyl pyrocarbonate-treated water. Total RNA was used for the detection of human ζ- and α-globin mRNA expression by RNase protection assay. In brief, 5 or 10 μg of total RNA was hybridized with 1–2 × 105 cpm of each [α-32P]-UTP labeled probe in solution at 55°C overnight. Then the samples were digested with RNase A (8 μg/ml) and RNase T1 (10 units/ml) at 25°C for 30 min. The protected fragments were purified and electrophoresed on a 4% polyacrylamide/7 M urea gel, the gel was exposed to an x-ray film for 24–28 h, and the band intensities were quantified by a scanning densitometer. The globin-specific probes were mouse α-globin, pT7mα; mouse ζ-globin, pT7 mζ (gifts from Qiliang Li, University of Washington, Seattle); human α-globin, pT7hα; and human ζ-globin, pSP6hζ (cloned by the National Laboratory of Medical Molecular Biology).
Results
Screening of BAC Library and FISH.
Ten candidate clones from the first panel of screening were determined further by Southern blot, only 191K2 and 465M8 clones had positive signals with HS-40 and θ-probes. After NotI digestion and pulse field gel electrophoresis of BAC DNA, the approximate sizes of the selected clones were estimated by comparison with size markers of multimers of bacteriophage λ (New England Biolabs). Clone 191K2 is 110-kb long and contains HS-40, ζ-, α-, and θ-globin genes. The physical map of the BAC 191K2 clone is shown in Fig. 1. The BAC 465M8 clone is 160-kb long but has only HS-40. Hence, clone 191K2 is the only one containing the whole human α-globin gene cluster and it was used for generating transgenic mice. FISH with this BAC showed that the hybridization signals were located at the telomere region of chromosome 16. No other signal was detected in the FISH results (Fig. 2).
Figure 1.
The physical map of BAC clone 191K2 of human α-globin gene cluster. Genes are indicated by the solid boxes, and pseudogenes are indicated by the open boxes. Arrows show the approximate positions of the hypersensitive sites. The positions of SalI and NruI restriction sites are indicated by vertical lines. The horizontal arrow represents another gene (14 gene or C16orf35) located on this cluster.
Figure 2.
FISH analysis of BAC clone 191K2. The arrows show the hybridization signals, which located at the telomere region of short arm of chromosome 16.
Generation and Identification of Transgenic Mice.
The 191K2 BAC DNA was purified as a linear 110-kb fragment and microinjected into fertilized eggs to generate transgenic mice. Genomic DNA purified from mouse tail was analyzed by PCR with HS-40 primers. Three founder mice were identified from 35 newly born mice by PCR (Fig. 3). Southern blot analysis was used to verify the PCR results. They all have the same hybrid bands with the human genomic DNA control (Fig. 4), which confirmed the structural integration of the human α-globin gene cluster. Compared with the human genomic DNA, the copy nos. for each strain of the three founders are 1 (no. 1), 2 (no. 2), and 2 (no. 3), respectively.
Figure 3.
PCR identification of transgenic mice. M, 200-bp DNA ladder; P, positive control; N, negative control; 1–3, transgenic founders. DNA samples were purified from transgenic mice tails by phenol/chloroform extraction, and about 0.5 μg of genomic DNA was amplified by PCR. Human genomic DNA was used as positive control, and mouse genomic DNA was used as negative control. The amplified fragment is about 500 bp.
Figure 4.
Southern analysis of transgenic mice. Purified genomic DNA (5 μg) of each sample was digested by PstI overnight at 37°C. Then the integrated BAC DNA was analyzed by Southern blotting, using probes of human HS-40, human α-globin, and ζ-globin fragments. Copy no. analysis was determined by phosphorimaging.
Expression of Human α- and ζ-Globin Genes in the Transgenic Mice Embryo.
The human α- and ζ-globin have very similar expression patterns with the mice endogenous ζ- and α-globin genes (Fig. 5). The human ζ-globin is switched off between days 10.5 and 12.5; meanwhile, the human α-globin is switched on. All of the three strains of transgenic mice have the same switching pattern. The expression level of human α-like globin gene decreased from 120–230% of human ζ-globin in embryo stage to 17–68% of human α-globin in adult stage (each copy), just like the results from cosmid transgenic mice. But we found that the human α-globin increased gradually after the switching and became stable after birth.
Figure 5.
Developmental stage-specific expression of human α-like globin genes in transgenic mice. RNA was extracted from individual 9.5- and 10.5-day yolk sacs of embryos and from fetal livers of individual 12.5- to 16.5-day fetuses. The probes used in the RNase protection assay were human α-pT7hα, ζ-pSP6hζ, mouse α-pT7mα, and ζ-pT7mζ.
Expression of Human α-Globin in Adult Transgenic Mice.
Fig. 6 showed the expression of human α-globin in mouse bone marrow. Compared with the mouse α-globin gene, the expression levels of human α-globin gene in adult transgenic mice are no. 1 40%, no. 2 17.5%, and no. 3 68% (each copy). The average level is about 42%, which is integration site-dependent.
Figure 6.
The expression of human α-like globin genes in adult transgenic mice. RNA was extracted from bone marrow of individual adult transgenic mice. The probes for the RNase protection assay were human α-globin and mouse α-globin genes.
Tissue-Specific Expression of Human α-Globin in Transgenic Mice.
The human α-globin had the very similar expression pattern of the mouse α-globin gene in different tissues of transgenic mouse no. 3. The human α-globin is expressed in mouse bone marrow, kidney, spleen, and brain, but not in muscle, testis, or thymus, indicating that the human α-globin gene expression is relatively erythroid-specific in transgenic mice. The relative expression level of human α-globin to the mice α-globin varied from tissue to tissue, 135% in the bone marrow and only 30% in the brain (Fig. 7).
Figure 7.
Tissue-specific expression of human α-like globin genes in transgenic mice. Bm, bone marrow; Br, brain; Ki, kidney; Li, liver; Mu, muscle; Sp, spleen; Te, testis; Th, thymus. Different tissues from one offspring of the No. 3 transgenic line were collected, and total RNA was extracted for the RNase protection assay.
Discussion
In the present study, we screened the human BAC library and obtained a 110-kb clone containing the whole human α-globin gene cluster. From the upstream regulatory element to the downstream sequence of the θ-globin gene, the BAC DNA was successfully introduced into mouse genome.
Based on the Escherichia coli F factor, the BAC vector has high stability and minimal chimerism and it can propagate up to 300 kb of foreign DNA, which is large enough to cover many gene clusters. Some research groups have generated several BAC-mediated large-fragment transgenic models (12, 13). Our results demonstrated that BAC can be used to perform research work with ease and reproducibility, even for gene clusters containing a high percentage of repeat sequences like the human α-globin gene cluster. Mutation analysis of BAC DNA will allow us to study the relationship between the structure and function of the entire gene cluster and may reveal other yet unknown regulatory elements that control transcription.
In our study, 3 founders of α-BAC transgenic mice from 35 newborn mice were detected by PCR and Southern blot analyses, which showed that the entire BAC DNA was integrated into the mice genome. The human α-globin displayed developmental stage- and relatively tissue-specific expression in the transgenic mice similar to the expression pattern of the mouse endogenous α-like globin genes. The expression level of the human embryonic ζ-globin is much higher than α-globin compared with the mice endogenous α-like globin genes in the yolk sac and fetal liver. The relative expression levels of human α-globin increased gradually during the switching and varied among the 3 lines from 17% to 68%, suggesting that the human α-like globin genes had integration site-dependent expression in transgenic mice. The expression pattern is different from the results of the BAC-mediated adult transgenic mice of human β-globin gene cluster in which human β-globin gene is expressed in similar levels in different transgenic lines (14). The β-globin locus is located at the middle of short arm of chromosome 11. The locus control region may be important for opening chromosome structure in erythroid cells. Hence, the expression of the human β-globin gene is not affected by the integration sites as we have previously found in four transgenic lines carrying the β-globin BAC (14). The human α-like globin gene cluster is located at the telomere region of chromosome 16, where the chromosome is in an open configuration in all tissues. The α-globin transgene may be integrated in the chromosome regions that are not in open configuration, resulting in variable expression of α-globin gene.
The mechanism for the different expression levels between human ζ- and α-globin in transgenic mice is not clear, maybe because of the different regulatory patterns between human ζ- and α-globin genes or between α-like globin genes. The promoter region of the human ζ-globin gene has more trans-acting factor-binding sites than that of human α-globin (15); therefore, the mouse DNA binding proteins could form a strong and stable transcriptional complex in this region and direct high-level expression of the human ζ-globin gene in transgenic mice.
A previous study showed that after the switching from ζ- to α-globin genes, the human α2- and α1-globin genes have equal expression levels in the fetal stage. Then the expression of α2-globin increases gradually and becomes dominant after birth (16). We did not find the “switching” from α1- to α2-globin in transgenic mice. In our results, the expression level of human α2-globin gene is higher than human α1-globin gene because of the switching from ζ-globin to α1/α2-globin (the ratio ranged from 1.83 to 3.2). Hence, there may be no regulatory factors in mice that can effect the expression level of the two human α-globin genes. There is only an 18-nt difference at the 3′ untranslating sequence of the mRNA (17, 18), thus the different expression level of the two α-globin genes might be related to their distance to HS-40 or the stability of their mRNA in human red blood cells.
Acknowledgments
We thank Dr. Qiliang Li (Univ. of Washington, Seattle) for providing pT7mα and pT7 mζ and Dr. James Shen for providing HS-40 and θ-probes. The work was supported by National Natural Science Foundation of China Grant 39893320 and by Science Foundation for Chinese Outstanding Youth Grant 3952006.
Abbreviations
- BAC
bacterial artificial chromosome
- FISH
fluorescence in situ hybridization
References
- 1.Liebhaber S A, Russell J E. Ann NY Acad Sci. 1998;850:54–63. doi: 10.1111/j.1749-6632.1998.tb10462.x. [DOI] [PubMed] [Google Scholar]
- 2.Turcinov D, Krishnamoorthy R, Janicijevic B, Markovic I, Mustac M, Lapoumeroulie C, Chaventre A, Rudan P. Coll Antropol. 2000;24:295–301. [PubMed] [Google Scholar]
- 3.Higgs D R, Wood W G, Jarman A P, Sharpe J, Lida J, Pretorius I M, Ayyub H. Genes Dev. 1990;4:1588–1601. doi: 10.1101/gad.4.9.1588. [DOI] [PubMed] [Google Scholar]
- 4.Vyas P, Vickers M A, Simmons D L, Ayyub H, Craddock C F, Higgs D R. Cell. 1992;69:781–793. doi: 10.1016/0092-8674(92)90290-s. [DOI] [PubMed] [Google Scholar]
- 5.Grosveld F, van Assendelft G B, Greaves D R, Kollias G. Cell. 1987;51:975–985. doi: 10.1016/0092-8674(87)90584-8. [DOI] [PubMed] [Google Scholar]
- 6.Craddock C F, Vyas P, Sharpe J A, Ayyub H, Wood W G, Higgs D R. EMBO J. 1995;14:1718–1726. doi: 10.1002/j.1460-2075.1995.tb07161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Sharpe J A, Wells D J, Whitelaw E, Vyas P, Higgs D R, Wood W G. Proc Natl Acad Sci USA. 1993;90:11262–11266. doi: 10.1073/pnas.90.23.11262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Bernet A, Sabatier S, Picketts D J, Ouazana R, Morle F, Higgs D R, Godet J. Blood. 1995;86:1202–1211. [PubMed] [Google Scholar]
- 9.Gourdon G, Sharpe J A, Wells D, Wood W G, Higgs D R. Nucleic Acids Res. 1994;22:4139–4147. doi: 10.1093/nar/22.20.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Green E D, Riethman H C, Dutchik J E, Olson M V. Genomics. 1991;11:658–669. doi: 10.1016/0888-7543(91)90073-n. [DOI] [PubMed] [Google Scholar]
- 11.Shizuya H, Birren B, Kim U J, Mancino V, Slepak T, Tachiiri Y, Simon M. Proc Natl Acad Sci USA. 1992;89:8794–8797. doi: 10.1073/pnas.89.18.8794. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Asakawa S, Abe I, Kudoh Y, Kishi N, Wang Y, Kubota R, Kudoh J, Kawasaki K, Minoshima S, Shimizu N. Gene. 1997;191:69–79. doi: 10.1016/s0378-1119(97)00044-9. [DOI] [PubMed] [Google Scholar]
- 13.Antoch M P, Song E J, Chang A M, Vitaterna M H, Zhao Y, Wilsbacher L D, Sangoram A M, King D P, Pinto L H, Takahashi J S. Cell. 1997;89:655–667. doi: 10.1016/s0092-8674(00)80246-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Huang Y, Liu D P, Wu L, Li T C, Wu M, Feng D X, Liang C C. Blood Cells Mol Dis. 2000;26:598–610. doi: 10.1006/bcmd.2000.0339. [DOI] [PubMed] [Google Scholar]
- 15.Sabath D E, Koehler K M, Yang W Q, Phan V, Wilson J. Blood Cells Mol Dis. 1998;24:183–198. doi: 10.1006/bcmd.1998.0185. [DOI] [PubMed] [Google Scholar]
- 16.Peri K G, Gagnon C, Bard H. Pediatr Res. 1998;43:504–508. doi: 10.1203/00006450-199804000-00011. [DOI] [PubMed] [Google Scholar]
- 17.Mamalaki A, Moschonas N. Acta Haematol. 1990;84:30–37. doi: 10.1159/000205023. [DOI] [PubMed] [Google Scholar]
- 18.Higgs D R, Sharpe J A, Wood W G. Semin Hematol. 1998;35:93–104. [PubMed] [Google Scholar]