MOLECULAR AND CELLULAR BIOLOGY,
`0270-7306/97/$04.0010
`Copyright q 1997, American Society for Microbiology
`
`Apr. 1997, p. 2076–2089
`
`Vol. 17, No. 4
`
`High Levels of Human g-Globin Gene Expression in Adult Mice
`Carrying a Transgene of Deletion-Type Hereditary
`Persistence of Fetal Hemoglobin
`MURAT O. ARCASOY,1 MARC ROMANA,1† MARY E. FABRY,2 EVA SKARPIDI,3
`RONALD L. NAGEL,2 AND BERNARD G. FORGET1*
`Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 065101; Department of
`Medicine, Albert Einstein College of Medicine, Bronx, New York 104612; and Division of Medical
`Genetics, University of Washington School of Medicine, Seattle, Washington 981953
`
`Received 18 October 1996/Returned for modification 1 December 1996/Accepted 21 January 1997
`
`Persistent expression of the g-globin genes in adults with deletion types of hereditary persistence of fetal
`hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3* breakpoints
`of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment
`containing both human g-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences
`of the locus control region (LCR) of the human b-globin gene cluster. Analysis of g-globin expression in six
`HPFH transgenic lines demonstrated persistence of g-globin mRNA and peptides in erythrocytes of adult
`HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric
`focusing showed the presence of hybrid mouse a2-human g2 tetramers as well as human g4 homotetramers
`(hemoglobin Bart’s). In contrast, correct developmental regulation of the g-globin genes with essentially absent
`g-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines,
`consistent with autonomous silencing of normal human g-globin expression in adult transgenic mice. Inter-
`estingly, marked preferential overexpression of the LCR-distal Ag-globin gene but not of the LCR-proximal
`Gg-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These
`findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint
`DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results
`indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated
`expression of the g-globin genes.
`
`The human b-like globin gene cluster is located on the short
`arm of chromosome 11 and contains five linked functional
`globin genes and an upstream locus control region (LCR). The
`tissue-specific expression of the embryonic, fetal, and adult
`globin genes is developmentally regulated with sequential ac-
`tivation and silencing of individual genes during ontogeny,
`characterized by two switches in the pattern of b-like globin
`gene expression during development. The first switch is ob-
`served early in development as the major site of hematopoiesis
`shifts from the embryonic yolk sac to the fetal liver and is
`characterized by the high-level expression of the fetal Gg- and
`Ag-globin genes that replaces the expression of the embryonic
`e-globin gene. A second switch, from g- tod - and b-globin gene
`expression, occurs late in gestation as well as in the perinatal
`period and results in high-level expression of the adult b-globin
`genes in the erythroid cells from the bone marrow, replacing
`the expression of the fetal g genes following birth (6, 9, 39, 44).
`The exact mechanisms that regulate globin gene switching are
`as yet incompletely understood. However, developmental reg-
`ulation of this multigene locus appears to be mediated by
`complex developmental stage- and tissue-specific interactions
`
`* Corresponding author. Mailing address: Yale University School of
`Medicine, Hematology Section, WWW 403, 333 Cedar St., New Ha-
`ven, CT 06510. Phone: (203) 785-4144. Fax: (203) 785-7232. E-mail:
`bernard.forget@yale.edu.
`† Present address: Unite´ de Recherche sur la Drepanocytose, IN-
`SERM U-359, Centre Hospitalier Universitaire, Guadeloupe, French
`West Indies.
`
`2076
`
`between cis- and trans-acting regulatory elements within the
`gene cluster.
`Genetic disorders that alter hemoglobin (Hb) switching have
`provided naturally occurring molecular models for the study of
`the regulation of globin gene transcription and the mecha-
`nisms of Hb switching during development. Naturally occur-
`ring deletions within the b-globin cluster that interfere with
`fetal-to-adult Hb switching result in two related but discrete
`clinical syndromes, db-thalassemia and hereditary persistence
`of fetal Hb (HPFH), which are characterized by inappropriate,
`persistent expression of the fetal g-globin genes in adult life.
`Individuals heterozygous for deletion-type HPFH have normal
`erythrocyte (RBC) parameters, but their Hb consists of 20 to
`30% Hb F which is distributed in a pancellular fashion among
`the RBCs. In contrast, individuals with heterozygous db-thalas-
`semia have smaller-than-normal RBCs and lower Hb F levels
`(10 to 15%), distributed in a heterocellular fashion (5, 34, 39).
`One hypothesis to explain persistent g-globin gene expression
`in adult life in these conditions is that the deletions remove
`sequences downstream of the g-globin genes which are neces-
`sary for the silencing of the genes in adult life (25). Another
`mechanism for persistent high levels of Hb F expression could
`involve the introduction into the vicinity of the g-globin genes
`of DNA sequences from the breakpoint regions that can influ-
`ence g-globin gene expression following birth through enhanc-
`er-like effects (1, 18, 35, 42). These two mechanisms are not
`mutually exclusive, however, and a different balance of these
`and possibly other effects could play a role in the different
`phenotypes of persistent g-globin gene expression observed in
`adults with HPFH and db-thalassemia.
`
`SKI Exhibit 2078
`Page 1 of 14
`
`

`

`VOL. 17, 1997
`
`DELETION-TYPE HPFH IN TRANSGENIC MICE
`
`2077
`
`FIG. 1. (A) Diagrams of the normal and mutant (HPFH-2) human b-globin loci. The arrowheads indicate the DNase I-HSSs in the LCR. (B) DNA fragments that
`were used to generate transgenic mice. See Materials and Methods for a detailed description of the DNA fragments used in microinjection. mLCR, mini-LCR cassette;
`Bg, BglII; H, HindIII; K, KpnI; S, SalI.
`
`A large body of experimental results has been generated by
`the analysis of a large number of different transgenic mice
`carrying a variety of fragments from the human b-globin gene
`cluster. In the absence of the LCR, individual human g-globin
`transgenes with short segments of flanking DNA have been
`shown to be expressed during mouse development in yolk
`sac-derived but not adult erythroid cells (7, 26). These early
`results suggested that DNA sequences necessary to mediate
`developmental stage-specific expression of the g-globin genes
`are located within or near the genes themselves. However,
`later experiments by Enver et al. (16) and Behringer et al. (3)
`showed that when linked with the LCR, g-globin transgenes in
`the absence of linked b-globin genes were expressed in both
`embryonic and adult RBCs and that silencing of g genes in
`adult erythroid cells was achieved by linking the LCR to the
`intact gdb region. These results suggested a competitive model
`for regulation of the g- and b-globin genes in transgenic mice
`(12, 15, 16, 23, 24). Subsequent experiments by Dillon and
`Grosveld (11) showed that g-globin genes linked to the LCR
`are in fact silenced autonomously in adult erythroid tissues in
`the absence of an adjacent competing b-globin gene. Experi-
`ments with transgenic mice carrying a 70-kb transgene contain-
`ing the entire human b-globin locus, including its LCR, in their
`normal structural configuration, as well as analyses of mice
`carrying the total b-globin gene cluster and LCR in large yeast
`artificial chromosome (YAC) transgenes, showed that the hu-
`man g-globin genes are correctly regulated during develop-
`ment and are expressed during the embryonic and early fetal
`
`stages of murine erythropoiesis (20, 32, 41). The phenotype of
`mice carrying large transgenes of the b-globin gene cluster but
`with deletions of both the d- and b-globin genes demonstrated
`autonomous silencing of the g-globin genes in adult transgenic
`mice, supporting the possible role of 39 breakpoint DNA se-
`quences in the generation of db-thalassemia and deletion-type
`HPFH phenotypes (33, 41).
`To investigate the molecular mechanisms of g-globin gene
`overexpression in adults with deletion-type HPFH, we have
`studied a model for the two most common forms of deletion-
`type GgAg pancellular HPFH, HPFH-1 and HPFH-2. These
`disorders are associated with extensive deletions of the b-glo-
`bin cluster involving approximately 105 kb of DNA (10) that
`includes both the d- and b-globin genes (Fig. 1A). The purpose
`of this study was to test the functional significance of the
`breakpoint DNA sequences in deletion-type HPFH and to
`determine whether the presence of the b-cluster LCR is re-
`quired for generation of the HPFH phenotype in transgenic
`mice carrying a 25-kb human DNA fragment mimicking the
`structure of the b cluster in HPFH-2. We generated two trans-
`genic lines carrying the HPFH-2 transgene without the LCR
`and six transgenic lines with the HPFH-2 transgene with the
`LCR. Analysis of the phenotypes of these transgenic lines
`indicates that adult mice carrying the HPFH-2 transgene with-
`out the LCR displayed a delay in the silencing of g-gene ex-
`pression during fetal development but did not have persistent
`g-gene expression in adult erythroid cells. On the other hand,
`analysis of the HPFH-2 transgenic lines with the LCR revealed
`
`SKI Exhibit 2078
`Page 2 of 14
`
`

`

`2078
`
`ARCASOY ET AL.
`
`MOL. CELL. BIOL.
`
`that human g-globin mRNA and peptide chains were persis-
`tently expressed at significant levels and incorporated into
`mouse a2-human g2 hybrid Hb tetramers. This persistent
`g-gene expression was associated with formation of a DNase
`I-hypersensitive site (HSS) within HPFH-2 39 breakpoint DNA
`region 3D in erythroid tissues of adult mice with the LCR/
`HPFH-2 transgene. In contrast, correct developmental regu-
`lation of g-globin gene expression was observed in animals
`from two different control non-HPFH transgenic lines carrying
`LCRGgAg and LCRGgAgcb transgenes in which there was
`appropriate silencing of the g-globin transgenes in adult ery-
`throid cells.
`
`MATERIALS AND METHODS
`
`DNA fragments. The following human DNA fragments shown in Fig. 1B were
`prepared for injection into mouse oocytes. (i) GgAg is a 13-kb BglII fragment
`containing both g-globin genes and downstream sequences up to approximately
`the 59 HPFH-2 breakpoint site. (ii) GgAgHPFH-2 is a 25-kb transgene contain-
`ing 12 kb of HPFH-2 39 breakpoint DNA sequences linked to GgAg. (iii)
`LCRGgAgHPFH-2 is an ;30-kb transgene containing a 4.7-kb LCR cassette
`linked to the 25-kb fragment. (iv) LCRGgAg is an ;18-kb transgene with the
`LCR cassette linked to the 13-kb BglII genomic DNA fragment. (v) LCRGgAgcb
`is an ;32-kb transgene containing the LCR cassette, both g-globin genes, and 14
`kb of downstream sequences ending 59 to the d-globin gene. The 13-kb BglII ge-
`nomic DNA fragment containing both the Gg and Ag genes (GenBank Humhbb
`coordinates: 32820 to 45698) was purified from a cosmid clone containing the
`bS-globin cluster described previously (8) and subcloned into the BamHI site of
`cosmid vector pWE15, which contained a modified polylinker. To construct the
`LCRGgAgHPFH-2 transgene, a 12-kb BglII-HindIII genomic DNA fragment
`from a l phage clone (lB-1) containing the normal DNA overlapping the 39
`breakpoint of HPFH-1 and HPFH-2 (42) was subcloned downstream of the
`13-kb BglII genomic fragment containing the g-globin genes to yield a 25-kb
`DNA fragment whose structure is virtually identical to the structure of HPFH-2
`DNA.
`An LCR cassette was constructed by ligating 59 HSS1 and HSS2 isolated as a
`from the mLAR plasmid (19) to a 1.2-kb
`1.4-kb PstI-HindIII fragment
`XmnI-HindIII fragment containing HSS3 and a 2.0-kb BamHI-XbaI fragment
`containing HSS4 (GenBank Humhbb coordinates: HSS1, 13062 to 13769; HSS2,
`8486 to 9218; HSS3, 3975 to 5172; HSS4, 308 to 2352). This 4.7-kb LCR cassette
`was subcloned in the genomic orientation upstream of the g-globin genes in the
`GgAgHPFH-2 DNA fragment to yield the LCRGgAgHPFH-2 transgene. The
`first control transgene, LCRGgAg, contained the LCR cassette, which was sub-
`cloned in the genomic orientation upstream of the 13-kb BglII genomic fragment
`containing the g-globin genes up to the HPFH-2 59 breakpoint. The second
`control transgene, LCRGgAgcb, was obtained by inserting the LCR cassette, in
`the genomic orientation, upstream of a 27-kb KpnI-SalI genomic fragment (Gen-
`Bank Humhbb coordinates: 27250 to 54726) containing the Gg- and Ag-globin
`genes and downstream sequences including the cb pseudogene up to the d-glo-
`bin gene.
`Transgenic mouse lines. The DNA fragments were prepared for microinjec-
`tion as described previously (40). A solution containing 10 ng of DNA per ml was
`injected into the pronuclei of (C57BL/6 3 SJL)F2 zygotes and transferred to
`pseudopregnant females. Transgenic mice were identified as described by Starck
`et al. (40), and transgenic founder mice were bred with nontransgenic B6/SJL
`mates to establish transgenic lines. Studies of transgene expression during de-
`velopment were performed with each transgenic line by mating hemizygous
`transgenic males with nontransgenic B6/SJL females to obtain embryos from
`timed pregnancies at 11.5, 13.5, and 16.5 days postcoitum. The morning on which
`the mating plug was observed was designated day 0.5. The copy numbers of the
`transgenes were determined by digestion of 10 mg of genomic DNA from the tail
`skin of progeny of two or three different animals from each line. Digestion with
`EcoRI, BamHI, or SacI was carried out and followed by 0.8% agarose gel
`electrophoresis and Southern blotting as described by Sambrook et al. (36).
`Several different probes from the transgenes were hybridized to the blots, and the
`hybridization signals were quantitated with a PhosphorImager (Molecular Dy-
`namics). The measurements were made several times, and averages were taken.
`Copy numbers were determined by comparing the signals from the human
`transgene to signals from mouse GATA-1 as described by Starck et al. (40).
`RNA analysis. Blood samples were obtained from mouse embryos and adult
`mice as described previously (40), and total RNA was isolated by using TRIzol
`reagent (Gibco-BRL) according to the manufacturer’s protocol. Human and
`murine globin mRNAs were analyzed by quantitative RNase protection assays.
`The human g-globin probe was a 403-bp NaeI-BamHI fragment (IVS1 deleted)
`in pGEM4 (Promega) containing 59 upstream sequences and exons 1 and 2 of the
`g-globin genes. The antisense probe was synthesized by transcription with Sp6
`polymerase to identify a 350-bp protected fragment. For the differential detec-
`tion of Gg- and Ag-globin transcripts, the Gg probe was transcribed with T7
`polymerase from pBluescript (Stratagene) containing a 721-bp HincII-HindIII
`
`Gg-globin fragment and the Ag probe was transcribed with Sp6 polymerase from
`pBluescript containing a 980-bp PvuII Ag-globin fragment, both of which con-
`tained the third exon including the 39 untranslated regions of the Gg- and
`Ag-globin genes, respectively, where four consecutive base differences are
`present between the Gg and Ag sequences beginning 3 bases 39 to the termina-
`tion codon. Both probes give a 215-bp protected fragment with the respective Gg
`or Ag transcript. Different riboprobes were used for the differential detection of
`Gg- and Ag-globin transcripts in the experiments with the mouse lines carrying
`the transgenes without the LCR. These probes and the riboprobes for detection
`of murine a-globin and z-globin mRNAs and quantitative mRNA analysis meth-
`ods used for the mouse lines carrying the transgenes without the LCR have been
`described previously (40). For the mRNA analysis of samples from transgenic
`mouse lines with the LCR, three different probes (human g-globin and mouse a-
`and z-globins) of 1.5 3 106 cpm total were simultaneously hybridized to 500 ng
`of total RNA from 11.5-day RBCs and 100 to 200 ng of RNA from RBCs of 13.5-
`and 16.5-day fetuses as well as newborn and adult mice. Samples were digested
`with RNases A (2.5 U/ml) and T1 (100 U/ml). Conditions of probe excess were
`confirmed in separate experiments. The protected fragments were detected by
`autoradiography after electrophoresis in 8% polyacrylamide–8-mol/liter urea
`gels. Quantitation of human and murine mRNAs was performed with a Phos-
`phorImager.
`Isoelectric focusing (IEF). Hb samples were separated on IEF by using a
`Resolve-Hb kit (Isolab, Akron, Ohio). Hb concentrations were adjusted to be-
`tween 2 and 4.5 g% by using the supplied sample preparation solution containing
`0.05% KCN. Gels were run until sharp bands formed, usually 2 h. To determine
`the percentage of each band on the IEF gel, the gel was fixed with 10% trichlo-
`roacetic acid solution; soaked in distilled water for 15 min; stained with the JB-2
`Staining System (Isolab) containing o-dianisidine, a heme-specific stain; and
`scanned at 520 nm with a model 710 densitometer (Corning, Medfield, Mass.).
`To identify bands, the band was removed from the unfixed gel, eluted with
`distilled water, spun to remove gel, and analyzed by high-performance liquid
`chromatography (HPLC).
`HPLC. The globin chain composition of mouse RBCs was determined by
`HPLC using a denaturing solvent that separates the globin chains and a Vydac
`large-pore (300-Å) C4 column (4.6 by 250 mm; Separations Group, Hesperia,
`Calif.) with a modified acetonitrile-H2O-trifluoroacetic acid (TFA) gradient sim-
`ilar to that used by Schroeder et al. (37) for separating human globin chains. Two
`buffers were used: A (0.18% TFA in 36% acetonitrile) and B (0.18% TFA in
`46% acetonitrile). Starting with 35% B, the amount of buffer B was increased by
`0.67%/min until all of the globin chains were eluted.
`Determination of oxygen affinity of hemoglobin (P50). Blood samples were
`collected from the mouse tail and placed directly into heparinized mouse saline
`(330 mosM). The samples were washed three times with 10 mM HEPES buffer
`containing 5 mM KCl, 5 mM glucose, and enough NaCl to adjust the osmolarity
`to 330 mosM. The buffer pH was 7.4 at 378C. O2 equilibrium curves were
`obtained by running the samples at 378C on a Hem-O-Scan O2 Dissociation
`Analyzer (Aminoco, Silver Spring, Md.) at hematocrits between 10 and 20%.
`DNase I hypersensitivity assays. Adult mice carrying the LCRGgAgHPFH-2
`transgene were made anemic by three injections of 0.2 ml of 0.4% acetyl phe-
`nylhydrazine per 25 g of body weight and sacrificed 5 days after the start of the
`injections to harvest the spleen. The spleen was placed in a 10-ml syringe and
`passed through an 18-gauge hypodermic needle several times and resuspended in
`RSB buffer (10 mM Tris z HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2). Intact
`nuclei were isolated as described elsewhere (43), and aliquots were subjected to
`DNase I digestion at various concentrations. The genomic DNA was purified by
`standard procedures (36). Appropriate restriction endonucleases were used to
`digest 15 mg of genomic DNA, and the digest was fractionated in 1% agarose
`gels. Southern blots were performed as described previously (36), and hybrid-
`ization was carried out with a probe from the breakpoint region known from
`previous experiments not to contain repetitive DNA sequences.
`
`RESULTS
`For studies of globin mRNA expression during develop-
`ment, F1 and F2 progeny of the founders were utilized and
`RBCs for mRNA analysis from multiple transgenic animals
`from each line were obtained at desired stages of development.
`For a given developmental stage in a transgenic line, RNA
`samples from multiple fetuses and adult animals were analyzed
`to minimize experimental error. Only adult animals 5 weeks
`old or older (range, 5 weeks to 2 years) were utilized in these
`studies to prevent any bias resulting from analyzing adult an-
`imals shortly after birth for g-globin gene expression.
`HPFH-2 3* breakpoint DNA sequences modify the develop-
`mental regulation of the g-globin genes in transgenic mice in
`the absence of the LCR. Two transgenic mouse lines carrying
`the GgAg transgene (lines 310 and 829) and two lines with the
`GgAgHPFH-2 transgene (lines 63 and 332) were established.
`
`SKI Exhibit 2078
`Page 3 of 14
`
`

`

`VOL. 17, 1997
`
`DELETION-TYPE HPFH IN TRANSGENIC MICE
`
`2079
`
`FIG. 2. (A) Human g-globin mRNA expression in RBCs of transgenic mice carrying the GgAgHPFH-2 transgene (line 332) and control (non-HPFH) GgAg
`transgene (line 310) without the LCR. Ten micrograms of total RNA isolated from mouse RBCs at each indicated developmental stage was hybridized to Gg- or
`Ag-globin riboprobes in RNase protection assays as described by Starck et al. (40). The expected position of the specific 161-nucleotide (nt) Gg or Ag protected RNA
`fragment is indicated. Total RNA from human K562 cells and nontransgenic mouse blood RNA (Control) served as positive and negative controls, respectively. (B)
`Quantitative representation of the ratios of human Gg or Ag mRNA to murine a plus z mRNAs at each developmental stage. Results of HPFH-2 line 332 are shown
`on the top, and results of non-HPFH line 310 are shown on the bottom. Expression of the murine a- plus z-globin mRNAs was quantitated in separate experiments
`using probes with different specific activities and 100 ng of total RNA in each hybridization (data not shown). Therefore, vertical-axis values should be regarded not
`as absolute values but as arbitrary units of the ratios of human to murine mRNAs. nb, newborn.
`
`Total RNA isolated from blood cells of mouse embryos at day
`11.5, from 13.5- and 16.5-day fetuses, and from newborn pups
`and adult animals was analyzed for the presence of human
`mRNAs. Autoradiographs of representative RNase protection
`assays of lines 332 (HPFH-2) and 310 (non-HPFH) are shown
`in Fig. 2A. Expression of Gg- and Ag-globin mRNAs was de-
`tected at day 13.5 in both HPFH-2 (line 332) and non-HPFH
`(line 310) lines. After day 13.5, however, the pattern of g-glo-
`bin mRNA expression during development differed signifi-
`cantly in HPFH-2 and non-HPFH mice. At day 16.5, levels of
`the two g mRNAs decreased markedly in mice carrying the
`GgAg transgene (Fig. 2A). In contrast, g mRNA expression is
`detected in 16.5-day fetuses as well as newborn mice carrying
`the GgAgHPFH-2 transgene (Fig. 2A). Quantitative analysis of
`these results is illustrated in Fig. 2B. The ratios of Gg- and
`Ag-globin mRNAs relative to murine z plus a mRNAs for each
`stage of development were determined. Because the level of
`expression of the murine globin genes is markedly greater than
`that of the human transgenes in the absence of the LCR,
`human and murine globin mRNAs were analyzed on different
`gels with different amounts of total cellular RNA as described
`by Starck et al. (40). Therefore, the quantitative data illus-
`trated in Fig. 2B represent not absolute values but relative
`arbitrary units of the ratio between human and murine globin
`gene expression at each stage of development. Similar results
`were obtained with lines 63 and 829 (data not shown). Thus,
`both Gg- and Ag-globin mRNAs were detected in RBCs of
`16.5-day fetuses as well as newborn pups in the two HPFH-2
`mouse lines, in contrast to the non-HPFH control mice carry-
`ing the GgAg transgene, where levels of g-globin mRNAs de-
`creased markedly by fetal day 16.5 and were not detectable in
`
`RBCs of newborn pups. Although no g-globin gene expres-
`sion was observed in RBCs of adult animals carrying the
`GgAgHPFH-2 transgene without the LCR, these preliminary
`results indicated that DNA sequences located 39 to the break-
`point of the HPFH-2 deletion could modify the developmental
`expression of the g-globin genes in transgenic mice and may be
`involved in the generation of the HPFH phenotype.
`The g-globin genes are persistently expressed in erythroid
`cells of adult transgenic mice carrying the LCRGgAgHPFH-2
`transgene. Because our initial experiments with transgenic
`mice carrying the GgAgHPFH-2 transgene without LCR
`showed g-globin gene expression later during development
`than control g transgenes but not in adult erythroid cells, we
`next wished to examine the effect of the LCR on g-globin gene
`expression and asked whether the LCR is required to repro-
`duce the full HPFH phenotype in transgenic mice. We ana-
`lyzed, by quantitative RNase protection assays, six transgenic
`lines carrying the 30-kb LCRGgAgHPFH-2 transgene for ex-
`pression of human g- and endogenous mouse a- and z-globin
`mRNAs during mouse development. An autoradiograph of a
`representative RNase protection assay of line 3012 is shown in
`Fig. 3. Following PhosphorImager quantitation, human g-glo-
`bin mRNA levels were expressed as a percentage of mouse a-
`and z-globin mRNA levels and then corrected for the copy
`number of the transgenes as determined by Southern blotting.
`The results of g-globin gene expression in the six transgenic
`mouse lines carrying the LCRGgAgHPFH-2 transgene are
`shown in Table 1. During the fetal stage of development, the
`highest level of human g-globin gene mRNA per transgene
`copy ranged from 7.3% 6 1.2% to 29% 6 6% of the total
`mouse a- plus z-globin mRNA level. In erythroid cells of all
`
`SKI Exhibit 2078
`Page 4 of 14
`
`

`

`2080
`
`ARCASOY ET AL.
`
`MOL. CELL. BIOL.
`
`FIG. 3. Human g-globin mRNA expression in HPFH-2 line 3012 carrying the LCRGgAgHPFH-2 transgene. Total RNA, isolated from blood of multiple individual
`animals at each developmental stage as indicated at the top of the figure, was hybridized simultaneously to human g-, mouse a-, and mouse z-globin riboprobes (see
`Materials and Methods for the specific riboprobes and RNA quantities used for hybridization). The expected positions of the specific 350-nucleotide (nt) (human g),
`157-nt (mouse a), and 151-nt (mouse z) protected RNA fragments are indicated. The positive control samples were total RNA isolated from mouse erythroleukemia
`cells (MELC) and human K562 cells. The negative (2) control samples were RNA extracted from blood of 13.5-day fetal and adult nontransgenic mice.
`
`adult mice carrying the LCRGgAgHPFH-2 transgene, persis-
`tent expression of g-globin mRNA was present, ranging from
`3.4% 6 0.2% to 8% 6 3.5% per transgene copy of endogenous
`mouse a-globin mRNA. Thus, as illustrated in Fig. 4, in adult
`animals, the expression of the g-globin genes declined to only
`about one-third the level of maximal expression during fetal
`development, with an average percentage of adult g mRNA
`level relative to the highest fetal g mRNA level of 34% 6 11%
`(range, 21 to 48%). The decline in the level of g-globin mRNA
`during development occurred predominantly prior to birth,
`with only a relatively minor decline after the newborn period,
`i.e., during the first 5 weeks following birth. The g-globin
`mRNA levels remained quite stable up to 2 years of age in all
`adult animals tested (data not shown). Treatment with phenyl-
`hydrazine did not have an effect on g-globin mRNA levels
`(data not shown). In all transgenic lines except line 3035, g-glo-
`bin gene expression was maximal at fetal day 11.5. Line 3035,
`which has 20 copies of the transgene, showed the highest g-glo-
`bin mRNA levels in 16.5-day fetuses in repeated experiments
`(Table 1). The results of g-globin gene expression in six dif-
`ferent transgenic lines carrying the LCRGgAgHPFH-2 trans-
`gene lead us to conclude that HPFH-2 breakpoint DNA se-
`quences can modify the developmental pattern of g-globin
`gene expression, resulting in significant levels of g-globin
`mRNA in adult RBCs. Since our experiments using the 25-kb
`GgAg-HPFH transgene without the LCR did not reveal per-
`sistent g-globin gene expression in adult erythroid cells, we
`
`further conclude that the LCR is required for generation of the
`HPFH phenotype in transgenic mice.
`Distribution of g-globin chains in RBCs was examined by
`staining of peripheral blood with monoclonal anti-g-chain an-
`tibodies. Figure 5 shows representative slides. The distribution
`of g-globin chains in adult RBCs was not pancellular, but
`overall a high percentage of the cells, ranging from 38 to 86%,
`stained positive.
`The g-globin genes are silenced in RBCs of adult transgenic
`mice carrying the control LCRGgAg and LCRGgAgcb trans-
`genes. To serve as controls for the experiments carried out with
`the HPFH transgenic mice, one transgenic line with the 18-kb
`LCRGgAg transgene and one line with the 32-kb LCRGgAgcb
`transgene were generated. We asked whether g-globin gene
`silencing could occur in the absence of a linked b-globin gene
`or HPFH-2 breakpoint DNA sequences in transgenic mice.
`Studies of gene expression during development by RNase pro-
`tection assays were carried out as described above. A repre-
`sentative autoradiograph of an RNase protection gel for the
`transgenic line carrying the LCRGgAgcb transgene is shown in
`Fig. 6. Table 2 summarizes the results of g-globin gene expres-
`sion during development for each control line. In the line
`carrying the control LCRGgAg transgene, the highest g-globin
`mRNA level during the fetal stage of development was 31% 6
`2.7% (per transgene copy) of mouse a- plus z-globin mRNA,
`and the level declined significantly to 0.4% 6 0.2% in adult
`animals. Similarly, in the line carrying the LCRGgAgcb trans-
`
`SKI Exhibit 2078
`Page 5 of 14
`
`

`

`VOL. 17, 1997
`
`DELETION-TYPE HPFH IN TRANSGENIC MICE
`
`2081
`
`Line
`(copy no.)
`
`3012 (4)
`
`3017 (2)
`
`3021 (9)
`
`3025 (4)
`
`3028 (4)
`
`3035 (20)
`
`TABLE 1. Globin gene expression in HPFH-2 transgenic mouse lines with the LCR
`
`Developmental
`age
`
`No. of
`animals
`
`% (mean 6 SD)
`
`Human g mRNA/(mouse
`a 1 z mRNA)a
`
`Human g mRNA/mouse
`a mRNA/copy
`
`Adult g mRNA/peak
`fetal g mRNA (%)
`
`11.5 days
`13.5 days
`16.5 days
`Newborn
`Adult (5–15 wk)
`
`11.5 days
`13.5 days
`16.5 days
`Newborn
`Adult (6–36 wk)
`
`11.5 days
`13.5 days
`16.5 days
`Newborn
`Adult (5–22 wk)
`
`11.5 days
`13.5 days
`16.5 days
`Newborn
`Adult (6–21 wk)
`
`11.5 days
`13.5 days
`16.5 days
`Newborn
`Adult (5–6 wk)
`
`11.5 days
`13.5 days
`16.5 days
`Newborn
`Adult (5–40 wk)
`
`3
`3
`3
`3
`6
`
`3
`2
`3
`3
`6
`
`3
`3
`3
`3
`8
`
`3
`3
`2
`3
`6
`
`3
`3
`3
`2
`4
`
`3
`2
`3
`3
`7
`
`86 6 6.5
`61 6 6.3
`65 6 6.3
`49 6 6.1
`31 6 6.0
`
`58 6 11
`49 6 13
`30 6 3.2
`15 6 3.4
`12 6 5.0
`
`79 6 24
`63 6 22
`58 6 1.6
`65 6 20
`38 6 11
`
`94 6 14
`58 6 6.6
`46 6 4.1
`39 6 5.5
`32 6 14
`
`65 6 7.0
`58 6 13
`38 6 3.2
`27 6 2.1
`15 6 3.9
`
`95 6 7.5
`132 6 2.9
`145 6 23.8
`159 6 14
`67 6 4.3
`
`22 6 1.6
`15 6 1.5
`16 6 1.6
`12 6 1.5
`7.8 6 1.5
`
`29 6 6.0
`25 6 6.5
`15 6 1.6
`8 6 1.7
`6 6 2.5
`
`9 6 2.6
`7 6 2.4
`6.4 6 0.2
`7.2 6 2.2
`4.2 6 1.2
`
`24 6 3.5
`15 6 1.6
`12 6 1.0
`9.8 6 1.4
`8 6 3.5
`
`16 6 1.7
`14 6 3.2
`9 6 0.8
`6.8 6 0.5
`3.8 6 0.9
`
`4.8 6 0.4
`6.6 6 0.1
`7.3 6 1.2
`8 6 0.7
`3.4 6 0.2
`
`36
`
`21
`
`48
`
`34
`
`24
`
`46
`
`a When only two animals were available, results of multiple quantitations from separate experiments were used to calculate means and standard deviations.
`
`gene, the g-globin mRNA level in RBCs of 13.5-day fetuses
`was 23% 6 2.9% (per transgene copy) of endogenous a- plus
`z-globin mRNA, compared to 0.3% 6 0.1% in RBCs of adult
`animals. Thus, g-globin gene expression in the adult stage of
`development from both control transgenic lines declined 70-
`fold, to 1.3% of maximal fetal-stage expression, compared to
`an only 3-fold decline, to 34% 6 11%, in the animals carrying
`the LCRGgAgHPFH-2 transgene (Fig. 4). Staining of adult
`RBCs of transgenic mice with anti-g-chain antibodies showed
`1% positive cells in the line carrying the LCRGgAgcb trans-
`gene (Fig. 5B) and 0.12% positive cells in the LCRGgAg line
`(data not shown). Although only two LCR-containing control
`lines were available for study, we conclude that g-globin trans-
`gene silencing in adult mice can occur in the absence of com-
`petition from the downstream d- and b-globin genes and that
`sequences required for this silencing effect reside within the
`LCRGgAg and LCRGgAgcb transgenes used in these experi-
`ments. Therefore, our results were similar to those obtained by
`Dillon and Grosveld (11) using LCR-g transgenes of somewhat
`different structure.
`IEF of Hb shows the presence of mouse a2-human g2 globin
`chain tetramers (a2
`Mg2) and g4 homotetramers. The Hb phe-
`notype of transgenic mouse RBCs was analyzed by IEF of pe-
`ripheral blood of adult animals carrying the LCRGgAgHPFH-2
`transgene, and the results were compared to those obtained
`
`from RBCs of nontransgenic animals and the control trans-
`genic lines carrying the LCRGgAg and LCRGgAgcb trans-
`genes. The adult animals were not treated with phenylhydr-
`azine prior to phlebotomy. IEF of blood samples from mice
`carrying the LCRGgAgHPFH-2 t