Proc. Natl. Acad. Sci. USAVol. 92, pp. 11608-1 1612, December 1995Genetics

A mouse model for 10-thalassemia(gene targeting/hemoglobin/splenomegaly/iron overload/extramedullary hematopoiesis)

BAOLI YANG*, SUZANNE KIRBY*, JADA LEWISt, PETER J. DETLOFF*T, NOBUYO MAEDA*, AND OLIVER SMITHIES*§*Department of Pathology, School of Medicine and tthe Curriculum in Genetics and Molecular Biology, University of North Carolina,Chapel Hill, NC 27599-7525

Contributed by Oliver Smithies, September 1, 1995

ABSTRACT We have used a "plug and socket" targetingtechnique to generate a mouse model of 130-thalassemia inwhich both the bi and b2 adult globin genes have been deleted.Mice homozygous for this deletion (Hbbth-3/Hbbth-3) die peri-natally, similar to the most severe form of Cooley anemia inhumans. Mice heterozygous for the deletion appear normal,but their hematologic indices show characteristics typical ofsevere thalassemia, including dramatically decreased hemat-ocrit, hemoglobin, red blood cell counts, mean corpuscularvolume, mean corpuscular hemoglobin, and mean corpuscularhemoglobin concentration, as well as dramatically increasedreticulocyte counts, serum bilirubin concentrations, and redcell distribution widths. Tissue and organ damage typical off3-thalassemia, such as bone deformities and splenic enlarge-ment due to increased hematopoiesis, are also seen in theheterozygous animals, as is spontaneous iron overload in thespleen, liver, and kidneys. The mice homozygous for the bi andb2 deletions should be of great value in developing therapiesfor the treatment of thalassemias in utero. The heterozygousanimals will be useful for studying the pathophysiology ofthalassemias and have the potential of generating a model ofsickle cell anemia when mated with appropriate transgenicanimals.

The a- and ,B-globin polypeptide chains of hemoglobin areencoded by multigene clusters that are generally conservedthroughout mammalian evolution. The mouse 3-globin genecluster on chromosome 7 (1) has four functional ,B-globingenes: O3hl, an early embryonic globin gene; sy2, a late embry-onic globin gene; and two adult globin genes, bl (/3major) andb2 ((3minor) (2). Mouse embryonic hemoglobins are first ex-pressed at 9.5 days of gestation in the yolk sac and later in fetalliver. The switch from predominantly embryonic to predom-inantly adult hemoglobins is completed between days 14 and15 of gestation in the normal fetus (3, 4). The bl and b2 genesare then expressed in fetal liver and spleen and, finally, in bonemarrow during adult life. The bi gene is reported to beresponsible for 80% and b2 for 20% of adult ,B-globinproduction (5).Human thalassemias are a heterogeneous group of inherited

disorders of hemoglobin synthesis, all characterized by theabsence of or reduction in amounts of one or more of theglobin chains of hemoglobin (6), which generates an imbalancebetween the subunits of hemoglobin. When the reduction isamong the adult ,B-globin genes, the resultant disorders aref3-thalassemias. These disorders can be divided into two majorgroups, (30 and f3+, based on the quantity of /3-globin produced;f31(-thalassemia results from the complete absence of the3-globin, and l3+-thalassemia results from a decrease in ,B-glo-

bin production.No naturally occurring 30-thalassemia has been observed in

mice, but two mouse models of f3+-thalassemia exist (7, 8). In

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

the first, a spontaneous DNA deletion of -3.7 kb in the,3-globin locus was found that includes the entire bl gene andits adjacent upstream sequences. Mice homozygous for thedeletion (Hbbth-l/Hbbth-l) survive to adulthood in 60% of thenumbers with wild-type (wt) mice. The heterozygous mice exhibita mild phenotype comparable to that of thalassemia trait inhumans (7). The second model for ,B-thalassemia was created bytargeted gene disruption of the mouse bl gene. Mice homozygousfor an insertional disruption of the bl gene (Hbbth-2/Hbbth-2) areseverely anemic and do not survive more than a few hours afterbirth (8). A possible explanation for the difference between thedeletion and disruption mouse models of thalassemia is thatthe non-globin-producing promoters in the disrupted /3-globinlocus can compete for access to the locus control regionneeded for transcription, thereby reducing transcription fromthe normal b2 gene (8). In the deletion animals, the b2 genehas no competitors. Similar differences in transcription havebeen observed in human thalassemic patients with deletions ormutations in the promoter region of the adult f-globin genecompared to patients with deletions or mutations that do notaffect the promoter region (9).The mice homozygous for deletion of both bi and b2 genes

that we have generated in the present study have a severe formof ,B0-thalassemia, similar to the most severe forms of Cooleyanemia in humans. The heterozygous animals show a patho-physiology comparable to that of patients with classical human,B-thalassemia intermedia.

MATERIALS AND METHODSAnimals. All the wt mice and mice heterozygous for the

,B-globin gene deletion studied here are F1 animals, exceptwhere stated otherwise. The F1 animals are offspring frommating between chimeras and mice of strain C57BL/6J (strainB6) and they have a hybrid genetic background derived fromthe two inbred strains: B6 and 12901a (strain 129). All micewere maintained in a room illuminated from 7 a.m. to 7 p.m.and were fed and watered ad libitum.Gene Targeting. Deletion of the mouse bl and b2 adult

,3-globin genes was accomplished by the two-step "plug andsocket" gene targeting strategy as described (10). Three inde-pendently targeted embryonic stem (ES) cells were injectedinto host B6 blastocysts (11). Chimeric mice generated fromtwo of them transmitted the targeted gene to their offspring.Mice heterozygous for the deletion have diagnostic bands of13.0 or 12.4 kb when genomic DNA is digested with Xba I orHpa I, Southern blotted, and probed with a 500-bp HPRT genefragment containing the HPRT promoter and the first exon(10).

Collection and Hematologic Analysis of Blood. Blood sam-ples were collected from the retroorbital sinus. For hemoglo-bin analysis, freshly drawn, heparinized blood samples werecollected, and the red cells were washed once in 20X vol ofphosphate-buffered saline and lysed in 5x vol of cystamine-

Abbreviations: ES cells, embryonic stem cells; wt, wild type.tPresent address: Department of Biochemistry and Molecular Genet-ics, University of Alabama, Birmingham, AL 35294.§To whom reprint requests should be addressed.

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containing buffer (12). Samples containing approximatelyequal amounts of hemoglobin were then run on celluloseacetate membranes (Helena Laboratories) (12). Hemoglobinbands were visualized by Ponceau S staining (Helena Labo-ratories).

For hematologic analyses, freshly drawn EDTA-treatedblood was used. Peripheral blood smears were stained withWright's stain (Sigma). Hematologic profiles were obtainedwith an automatic blood cell counter (Roche Cobas HeliosHematology Analyzer), which gives complete blood cell counts.Reticulocyte counts following supravital staining with Ac-custain (1% new methylene blue/0.9% sodium chloride/1.6%potassium oxalate) (Sigma) were determined by standardmethods (13). Serum bilirubin concentrations (total and di-rect) were determined on freshly collected blood using a

reagent kit (procedure no. 605) from Sigma.Scanning electron microscopy was performed on freshly

drawn, heparinized blood samples. After washing once withsaline, packed red blood cells were fixed with glutaraldehydeand allowed to settle on polylysine-coated glass coverslips.Blood cells were then dehydrated in a graded series of ethanoland critical-point dried. After drying, blood cells were coatedwith carbon followed by a 60:40 mixture of gold/palladium forviewing with a Cambridge scanning electron microscope at 10kV.

Histology. Mice for histologic studies were sacrificed with a

lethal dose of tribromoethanol (Avertin). The abdominal andthoracic cavities were opened and visceral organs were per-fused through heart puncture with phosphate-buffered para-formaldehyde (4%; pH 7.4) under physiological pressure.Spleen, liver, heart, lungs, and kidneys were removed, weighed,embedded, and sectioned. Specimens were stained with he-matoxylin and eosin and iron stains (Prussian blue).

Skulls and left femora from wt and heterozygous animalswere removed, decalcified, embedded, and sectioned for his-tologic assessment by hematoxylin and eosin staining.

Statistical Analysis. The two independent samples t test wasused (SYSTAT; see ref. 14) for comparing hematologic valuesbetween wt and heterozygous mice.

RESULTS AND DISCUSSIONGeneration of ,3-Thalassemic Mice. In an earlier study from

our laboratory, Detloff et al. (10) generated two targeted EScell lines that had both the mouse adult ,B-globin genes (bi andb2) deleted. The deletion plug P3Aplugl.5 was also reinsertedinto the socket-containing ES cell line B20 as described (10),and Southern blot analysis revealed the presence of the desireddeletion in one of six hypoxanthine/aminopterin/thymidine-resistant colonies. The three targeted ES cell lines were usedto generate chimeric animals by injection into host blastocysts.Of 10 chimeras generated from two of the three targeted EScell lines, two males and two females transmitted the ES cellgenome to their offspring after being mated to mice of strainB6. The resulting (B6 x 129)F1 hybrids were initially geno-typed by Southern blot analysis for the bl and b2 globin genedeletion using genomic DNA from tail biopsies. Subsequently,heterozygotes were genotyped by blood smears, since theirsmears were so different from those of wt mice.

Body Weight Difference. The F, heterozygotes at the time ofbirth are pale and small in comparison with their wt litter-mates. At weaning (3 weeks), significant differences (P <0.005) in body weight between F1 heterozygotes and male orfemale F1 wt animals were observed (Table 1). Body weightdifferences between wt F2 and heterozygous F2 pups at wean-ing are smaller although still significant (P < 0.05, if all mice,both male and female, are compared; see Table 1). Bodyweight differences in the F, mice decreased with age, becomingstatistically insignificant by 8-10 weeks of age (data notshown).Homozygous Animals. Eight intercrosses between F1 het-

erozygotes yielded 45 pups and 5 stillborn animals. Sevenlive-born pups were extremely pale and died within 12 hr. All7 mice dying within 12 hr and the 5 stillborn ones were

determined to be homozygous for the bi and b2 deletions. Theoverall numbers of each genotype were not statistically differ-ent from Mendelian expectations (15 wt, 23 heterozygotes, and12 homozygotes; P 0.7; x2 analysis). The mice homozygousfor the bi and b2 deletions resemble human patients with thetransfusion-dependent homozygous forms of ,B0-thalassemiathat are lethal in the untreated state. They differ from humansin that they die within hours after birth rather than later inchildhood. The increased severity of the phenotype in miceprobably occurs because the genetic switch from embryonic toadult hemoglobin occurs 7-8 days before birth in mice. Inhumans, the switch from synthesizing fetal to adult hemoglobinis not completed until several months to a year after birth, anduntransfused human f311-thalassemia homozygotes die in lateinfancy (15).Red Cell Morphology. Blood smears (data not shown) and

scanning electron micrographs (Fig. 1) were obtained using theblood of wt and heterozygous mice 7 weeks of age. Theheterozygotes show characteristics typical of those seen inpatients with severe thalassemia. There is marked anisocytosisand poikilocytosis; there are many microcytes, occasionalpolychromatic macrocytes, and frequent target cells. The redcells are generally hypochromic with variable numbers ofteardrop and oval forms, and fragmented cells are seen.

Nucleated red blood cells are also present. Pappenheimerbodies from precipitated a-globin chains are seen in the redblood cells.

Hematologic Studies. Hematologic studies included mea-

surements of total hemoglobin, red blood cell count, hemat-ocrit, reticulocyte count, and serum bilirubin concentrations(total and indirect) on wt and heterozygous mice 6-7 weeks ofage. A summary of the results is shown in Table 2. Miceheterozygous for the deletion are anemic, as is reflected in themarkedly decreased hematocrit, hemoglobin concentration,and red blood cell count. Their reticulocyte counts are dra-matically increased (21-fold), indicating a state of activeerythropoiesis in the heterozygous animals. The elevatedserum concentrations of total and indirect bilirubin in het-erozygous animals indicate an increased rate of red blood cellturnover and hemolysis. The high reticulocyte counts, low redblood cell counts, and increased indirect bilirubin in heterozy-gous mice indicate that both production and destruction oferythrocytes are increased in heterozygous compared to wtmice.

Table 1. Body weight of weanlings

F1 cross F2 cross

All mice Male Female All mice Male Female

wt 11.8 + 0.4 (n = 24) 11.7 + 0.6 (n = 14) 12.0 + 0.4 (n = 10) 9.2 + 0.3 (n = 47) 9.1 + 0.3 (n = 31) 10.1 + 0.5 (n = 16)Heterozygotes 9.0 + 0.6 (n = 18) 8.7 + 0.7 (n = 10) 9.4 ± 0.6 (n = 8) 8.5 + 0.2 (n = 39) 8.5 ± 0.3 (n = 23) 8.4 ± 0.3 (n = 16)P 0.00001 0.003 0.001 0.044 0.218 0.009

Body weights are expressed as means + SEM.

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FIG. 1. Scanning electron micrograph of blood cells from a heterozygous F1 13-thalassemic mouse and a wt littermate at 7 weeks of age. (A)wt mouse. (B) Mouse heterozygous for bl and b2 deletions. (X5390.)

Human thalassemic patients have a characteristic hypochro-mic, microcytic anemia, with decreased mean corpuscularvolume, mean corpuscular hemoglobin, and mean corpuscularhemoglobin concentration and increased red cell distributionwidth (15). Mice heterozygous for the bl and b2 gene deletionshave similar changes (Table 2) in all these parameters.Hemoglobin Electrophoresis. The absence of 13-globin pro-

duction from the altered strain 129-derived ,B-globin locus wasestablished by studying animals heterozygous for a normal3-globin locus that directs synthesis of a distinguishable formof adult ,3-globin. Hemoglobin from F1 heterozygous animalshave a wt (HbbS haplotype) chromosome from B6 and thestrain 129-derived deletion chromosome, which would nor-

mally direct the synthesis of major and minor ,B-globin (Hbbdhaplotype). When cystamine-modified hemoglobins are sepa-rated by cellulose acetate electrophoresis (12), wt (B6 x129)F1 mice have the B6-derived single and the 129-derivedmajor and minor ,B-globin (Fig. 2, lane 2). The heterozygousthalassemic mice have only the B6-derived single ,B-globin(lane 3), verifying that the deletion chromosome contributedno adult ,B-globin chains (5).

Pathology and Histology. Pathologic changes revealed bygross and microscopic examination of the heterozygous ani-mals were similar to those of human patients with severe

thalassemia. Spleen, liver, heart, lung, and kidney weights weredetermined as ratios to total body weight for both wt andheterozygous mutant animals. There were no differences inthese ratios for liver, heart, lungs, and kidneys between wt andheterozygous animals. There was, however, a highly significantincrease (P < 0.001) in the spleen weight/body weight ratiofrom heterozygous animals compared to wt control mice (2.80± 0.24 mg/g for wt mice versus 21.61 ± 4.54 mg/g forthalassemic mice). Splenomegaly is a common feature inhuman thalassemia patients.

Histologic studies of the heterozygotes also revealed signif-icant changes. Again the most dramatic changes appeared tobe in the spleens of the animals due to increased extramed-ullary hematopoiesis (Fig. 3). Spleen sections of wt mice showthe typical normal histology with distinct red and white pulpin approximately equal proportions, with the white pulp-containing follicles having germinal centers and the red pulphaving hematopoietic cells. Megakaryocytes are present in thered pulp, and trabeculae can be seen. In the spleens from miceheterozygous for the bl and b2 deletions, there is no cleardistinction between red and white pulp. Instead, the white pulpis almost completely replaced by hematopoietic tissue, andthere is impressive erythroid hyperplasia. Occasional germinalcenters can be seen perivascularly. More megakaryocytes are

Table 2. Hematologic values

RBC, MCV, MCHC, Bilirubin, mg/dlHct, % Hb, g/dl 106/mm3 Retics, % gm3 MCH, pg g/dl RDW Total Indirect

wt F1 45.7 ± 0.8 16.5 ± 0.5 9.5 ± 0.4 1.4 ± 0.4 51.1 ± 0.2 18.6 ± 0.2 365 ± 0.4 17.4 ± 0.6 0.18 ± 0.07 0.14 + 0.03(n = 30) (n = 28) (n =27) (n =5) (n = 18) (n = 18) (n = 18) (n = 18) (n =10) (n =10)

Heterozygous F1 30.5 ± 0.8 10.9 ± 0.3 6.6 ± 0.1 30.3 ± 0.9 43.0 ± 0.5 17.6 ± 0.4 30.7 ± 0.4 46.1 ± 0.5 0.45 ± 0.06 0.36 + 0.07(n = 28) (n = 28) (n =24) (n =4) (n = 17) (n = 17) (n = 17) (n = 17) (n =7) (n =7)

P <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 0.014 <0.00001 <0.00001 0.00004 0.00004% changes -33 -34 -31 2064 -16 -5 -16 165 150 157

Hematologic 'values are expressed as means ± SEM. Hct, hematocrit; RBC, red blood cells; Retics, reticulocytes; MCV, mean corpuscular volume;MCH, mean corpuscular hemoglobin; MCHC, MCH concentration; RDW, red cell distribution width.

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12 3 4

_- Origin

- Hbbd. minor.,o - Hbbd, mlaor- Hbbs

FIG. 2. Cellulose acetate electrophoretic analyses of mouse hemo-globins. Cystamine-modified hemoglobin was electrophoresed on cel-lulose acetate gel. Lanes 1-4, hemoglobins from wt mice of strain B6,wt F1 mice, F1 mice heterozygous for bl and b2 deletions, and wt miceof strain 129, respectively.

also present. Iron stains of spleen sections from wt mice revealonly trace amounts of iron in macrophages, while the thalas-semic mice have abundant, heavily stained iron particles inwhat appear to be "nurse cells," the macrophages in the centerof an area of erythropoiesis.

In both the bone and bone marrow of skulls and femora,significant differences were seen between wt and heterozygousmice. wt mice have thicker cortical bone in their skulls than doage-matched thalassemic mice. The femora of wt mice alsohave a shorter zone of proliferation and a longer zone ofosteogenesis compared to those from thalassemic mice; thismay contribute to the growth retardation seen in thalassemicmice. Relative to wt, mice heterozygous for the deletion haveincreased bone marrow cellularity as well as increased numbersof megakaryocytes and erythroid precursors.

In the older animals (5-6 months), histologic studies of theheterozygous and wt animals show that the spleen is still themajor affected organ. The histologic differences are similar tothose seen in younger animals, except that splenic iron depositsare significantly increased in the thalassemic mice, and irondeposits are now observed in both red and white pulp. Thethalassemic mice have abnormal iron accumulation in theproximal convoluted tubules of their kidneys. This is not seenin wt mice and is indicative of hemolysis comparable to thatseen in human thalassemic patients. Liver iron stores/deposits,which are minimal in wt animals, are abundant in both thehepatocytes and Kupffer cells of thalassemic animals (Fig. 4).

Iron Deposits. Our study provides clear evidence of spon-taneous iron deposition in the 03-thalassemic mice. Spontane-ous iron overload in nontransfused human thalassemia pa-tients arises not from an intrinsically flawed iron regulation orfrom an excess of dietary iron but rather from a response to theabnormal stimulation of increased erythropoietic activity (16-18).

Iron deposition is evident in mice heterozygous for the bland b2 deletions, even at a young age (4-5 weeks), in bothlivers and spleens. Iron accumulation in these organs was

a Tb,~~,,,C .

FIG. 3. Histology of spleens from a heterozygous F1 f3-thalassemicmouse and a wt littermate. Hematoxylin and eosin staining of spleenfrom a 6-week-old wt mouse (a) and a 6-week-old mouse heterozygousfor the bi and b2 deletions (b). (x100.)

progressive. Garrick et al. (17) observed that iron depositionincreases with age until plateaus in iron density and total ironcontent are reached at 200 days of age in Hbbth-l homozygotes.Raja et al. (18) found that liver iron deposition increases inHbbth-I homozygotes until the age of 7-8 weeks, but it showsno further increase over successive weeks.

Hemosiderosis is the result of translocation of iron from thehemoglobin of red cells into the cells of the reticuloendothelialsystem in liver, spleen, and bone marrow. Normally, siderosisis innocuous and reversible and is even seen as a seasonalphenomenon in some animal species (19). However, contin-uous accumulation of iron in liver parenchymal cells can havedeleterious effects. Although in humans iron overload occursmainly in hypertransfused thalassemic patients, it can occur inyoung thalassemic patients before any transfusional therapy(20, 21). In these individuals, iron particles are mainly seen inperiportal liver parenchymal cells.Comparison of Mouse Models of 3-Thalassemia. Mice

heterozygous for the bi and b2 deletions (which we propose todesignate Hbbth-3) have similar, although more severe, abnor-malities in their hematologic parameters and pathologic andhistologic findings to those of mice homozygous for the Hbbth-Imutation (7). Mice homozygous for insertional disruption of thebi gene (Hbbth-2/Hbbth-2) have a more severe phenotype thanthat of the heterozygous Hbbth-3/Hbbs mice or than that ofHbbth-l/Hbbth-l mice homozygous for the bi deletion (8). Therelative severities of the three conditions are of some interest.The severity of f3-thalassemia is directly related to the imbal-ance in synthesis between a- and 13-globins and so to the excessamount of a-globin produced (22). In normal strain 129 mice,there are two bi and two b2 genes; the two b2 genes produce-20% of all ,B-globin chains (5). In the absence of anycompensatory and competitive effects, mice homozygous forthe Hbbth-l bi deletion or the Hbbth-2 bl disruption would beexpected therefore to have only 20% of normal ,B-globin

FIG. 4. Histology of spleen, kidney, and liver from F1 heterozygous 3-thalassemic mice. Iron staining of spleen (a), kidney (b), and liver (c)from a 5-month-old F1 mouse heterozygous for the bi and b2 deletions. Dark stained particles within cells are iron deposits. (X400.)

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production, while mice heterozygous for the Hbbth-3 bi and b2deletions would be expected to have -50% of normal ,B-globinsynthesis. However, in practice mice homozygous for theHbbth-I mutation (which have only the two adult b2 genes)produce more ,B-globin than expected (-75% of normal ratherthan the expected 20%) (23), while mice homozygous for theHbbth-2 mutation do not survive and probably produce less,B-globin than the expected 20% (B. M. Brunner-Jackson,W. R. Shehee, O.S., and J. B. Whitney, unpublished data).These altered expressions have been attributed to translationalcompetition between a- and ,B-globin mRNAs and/or tran-scriptional competition for access to the locus control regionamong all promoters (globin and nonglobin) in the ,B-globinlocus (8, 24). In Hbbth-l/Hbbth- mice, the b2 gene promoter hasno competitor, while in Hbbth-2/Hbbth-2 mice the b2 genepromoter has both the neo gene promoter and the nonpro-ductive bl gene promoter as competitors. In our Hbbth-3 mice,with the deletion of both bi and b2 genes, any compensatoryand/or competitive factors within the 13-globin gene locus areeliminated.

CONCLUSIONMice heterozygous for the Hbbth-3 deletion generated in thisstudy have a similar yet more severe phenotype than micehomozygous for the Hbbth-l deletion or heterozygous for theHbbth-2 insertion. The Hbbth-3 heterozygotes have anemia,splenomegaly, and bone abnormalities similar to those ofhuman f30-thalassemic heterozygotes. However, unlike mostuntransfused human thalassemics, the animals develop spon-taneous iron deposits very early. The animals should conse-quently be-useful not only for studying the pathophysiology ofthalassemias but also for studying iron overload in differentorgans. They have the potential of being useful for testing newways of treating these diseases and the accompanying ironoverload. In addition, the heterozygotes will be useful formating with appropriate transgenic animals to generate apotentially improved mouse model of sickle cell anemia. Micehomozygous for the Hbbth-3 deletion may be valuable indeveloping in utero therapies for the treatment of severe andlethal forms of thalassemia.

Note Added in Proof. After submission of this manuscript, a reportdescribing similar work was published (25).

We thank B. Bagnell, K. Kluckman, V. Madden, and D. Reams fortechnical assistance. We also thank Barry Whitney (Medical Collegeof Georgia) and Donna King (Chicago Medical School) for their

Proc. Natl. Acad. Sci. USA 92 (1995)

critical reading of our manuscript. This work was supported in part byNational Institutes of Health Grants HL37001 and GM20069 to O.S.

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