Pre-B cell proliferation and lymphoblasticleukemia�high-grade lymphoma in E�-miR155transgenic miceStefan Costinean, Nicola Zanesi, Yuri Pekarsky, Esmerina Tili, Stefano Volinia, Nyla Heerema, and Carlo M. Croce*

Comprehensive Cancer Center, Ohio State University, 400 West 12th Avenue, Columbus, OH 43210

Contributed by Carlo M. Croce, March 22, 2006

MicroRNAs (miRNAs) represent a newly discovered class of post-transcriptional regulatory noncoding small RNAs that bind totargeted mRNAs and either block their translation or initiate theirdegradation. miRNA profiling of hematopoietic lineages in humansand mice showed that some miRNAs are differentially expressedduring hematopoietic development, suggesting a role in hemato-poietic cell differentiation. In addition, recent studies suggest theinvolvement of miRNAs in the initiation and progression of cancer.miR155 and BIC, its host gene, have been reported to accumulatein human B cell lymphomas, especially in diffuse large B celllymphomas, Hodgkin lymphomas, and certain types of Burkittlymphomas. Here, we show that E�-mmu-miR155 transgenic miceexhibit initially a preleukemic pre-B cell proliferation evident inspleen and bone marrow, followed by frank B cell malignancy.These findings indicate that the role of miR155 is to inducepolyclonal expansion, favoring the capture of secondary geneticchanges for full transformation.

transgenic mouse � malignant lymphoproliferation � microRNAs

M icroRNAs (miRNAs) represent a new class of abundantsmall RNAs that play important regulatory roles at the

posttranscriptional level; by binding to their targeted mRNAs,they either block their translation or initiate their degradation,according to the degree of complementarity with the target.

Since their discovery in 1993 in Caenorhabditis elegans (1),there have been numerous reports that implicated these tinymolecules in the posttranscriptional regulation of a large arrayof proteins with very diverse roles, ranging from cell prolifera-tion and differentiation to lipid metabolism (2–6).

miRNA profiling of hematopoietic lineages in humans andmice showed that miRNAs are differentially expressed in thecourse of hematopoietic development, suggesting a potentialrole in hematopoietic differentiation (7–9). We have shown thatmiR-15a and miR-16-1 are deleted or down-regulated in �68%of cases of chronic lymphocytic leukemia (CLL) (10, 11), andthat miRNAs genes are frequently located at fragile sites andother genomic regions involved in cancers (12). Transcripts ofmiR155 and BIC (its host gene) transcripts have been shown toaccumulate in human B cell lymphomas, especially diffuse largeB cell lymphomas (13), Hodgkin lymphomas (14), and subsets ofBurkitt lymphomas (latency type III Epstein–Barr virus-positiveBurkitt lymphoma; ref. 15). These reports provide indirectevidence that miR155 may play a role in B cell development andlymphomagenesis. We have also reported that miR155 is over-expressed in the aggressive form of CLL (11).

Here, we show that the transgenic mice carrying a miR155transgene whose expression is targeted to B cells (E�-mmu-miR155) exhibit initially a preleukemic pre-B cell proliferation,evident in spleen and bone marrow, and later develop a frank Bcell malignancy.

Results and DiscussionProduction and Characterization of E�-mmu-miR155. We generatedtransgenic mice in which the expression of mmu-miR155 (mouse

miR155) is under the control of a VH promoter-Ig heavy chainE� enhancer, which becomes active at the late pro-B cell stageof the B cell development. Fifteen transgenic founders wereidentified by Southern blot hybridization, seven on C57BL�B6and eight on FVB�N backgrounds. These were bred to wild-typemice of the same strain to produce 15 independent transgeniclines.

Northern blot and real-time PCR analysis (data not shown)performed on total RNA extracted from transgenic and wild-type spleens showed a very good expression of miR155, de-scribed in Fig. 1c. All other founder lines but one also expressedthe transgene. Wild-type mice did not express mature miR155 inthe spleen, as reported previously (16).

E�-mmu-miR155 Exhibited Splenomegaly as Early as 3 Weeks of Age.Spleens of transgenic mice were enlarged, with a spleen weight�body weight ratio three to four times greater than that ofwild-type mice at 3 weeks of age and did not increase signifi-cantly with age (Table 1 and Fig 2).

Transgenic Mice Overexpressing miR155 Are Leukopenic. The whiteblood cell count (WBC) of transgenics 3 months of age was 10 �106 � 1 � 106�ml compared with 40 � 106 � 1.5 � 106�ml fornormal age-matched mice. The WBC for transgenic mice 6months of age was even lower, at 6 � 106 � 0.5 � 106�ml,whereas that of wild type was unchanged.

Histological and Immunohistochemical Analysis of E�-mmu-miR155Mice. The histopathology of the spleens [hematoxylin�eosin(H&E) stain] from 3-week-old mice featured a consistent atyp-ical lymphoid population invading the red pulp and expanding it;the lymphoid follicles were unaffected, and there were multiplefoci of secondary hematopoiesis (Fig. 3a). Mice at 6 months ofage presented histologically a greatly increased malignant lym-phoid population with marked atypia and blastic appearance,proliferating in the vascular channels of the red pulp andgradually replacing the white pulp. The number of lymphoidfollicles was decreased, and the overall architecture of the spleenwas distorted by lymphoid proliferation (Fig. 3 b and c). Ahistologically similar lymphoid population was present in thebone marrow of 6-month-old mice. Expression of the prolifer-ation Ki67 antigen showed a marked lymphoid proliferation intransgenic mice, not observed in wild-type mice (Fig. 3f).

Immunohistochemistry performed to identify the immuno-phenotype of the lymphoid proliferation showed low positivityof the atypical expanded lymphocytes for B220 and VpreB1(CD179a), although CD43 was negative (data not shown). TheIgM staining of the paraffin-embedded sections of the trans-genic spleens showed the presence of � chains in the cytoplasm

Conflict of interest statement: No conflicts declared.

Abbreviations: H&E, hematoxylin�eosin; miRNA, microRNA; WBC, white blood cell count.

*To whom correspondence should be addressed. E-mail: croce.5@osu.edu.

© 2006 by The National Academy of Sciences of the USA

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of the proliferating lymphocytes (Fig. 4), whereas f low cytom-etry analysis failed to identify the expression of IgM on thesurface of these cells, indicating that the expanded lymphoidcells expressed cytoplasmic � chain but did not expresssurface IgM.

Flow Cytometry Analysis of E�-mmu-miR155 Reveals an Expansion ofthe B220low�CD10low�IgM��CD5��TCR��CD43� Population. Flow cy-tometry analysis, performed on single-cell suspensions of WBCfrom spleens and bone marrows of transgenic and wild-type miceof 3, 6, and 7 weeks and 6 months of age, showed an increase ofB220low�CD10low�IgM��CD5��TCR��CD43� lymphoid cellsin both spleen and bone marrow in the transgenic mice com-pared with their wild-type counterparts. This phenotype resem-bles the phenotype of proliferating lymphocytes observed inhuman acute lymphoblastic leukemia or lymphoblastic lym-phoma. Our findings indicate that the B220low�CD19low�CD10low�IgM��TCR��CD43� lymphoid population in the

Fig. 1. Production and characterization of E�-mmu-miR155. (a) A construct for the miR155 transgene was designed as shown. The mmu-miR155 was clonedbetween the EcoRV and SalI sites, putting the transgene under the control of the VH promoter E� enhancer. The construct was then injected in the male pronucleiof the oocytes of pregnant C57BL�6 and FVB�N female mice. (b) Southern blot was used to genotype the founders. Fifteen transgenic founders were born, sevenon a C57BL�6 background (b Left, lanes 1, 3, 5, 6, 7, 10, and 14 are transgenics, and lanes 2, 4, 8, 9, 11, 12, 13, and 15 are wild types) and eight on an FVB�Nbackground (b Right, lanes 1, 3, 5, 7, 9, 11, 13, and 15 are transgenics, and lanes 2, 4, 6, 8, 10, 12, and 14 are wild types). These were then bred to wild-typestrain-matched mice to produce 15 transgenic lines. (c) In each of the 15 transgenic lines, expression of the transgene was assessed by Northern blot on total RNAextracted from the lymphocytes isolated from the spleens of 3-week-old mice by using the antisense oligonucleotide of the mmu-miR155 mature sequence asa probe. Five of the transgenic lines with the highest level of expression of the mature miR155 in the splenocytes were selected for further breeding and analysis;one transgenic line did not express the transgene [lanes 1, 2, 3, 5, 8, and 9 (transgenics); lanes 4, 6, and 7 (wild types)].

Table 1. Spleen and body measurements for transgeniccompared with wild-type mice

MiceLine

(founder)Age,wks

BW,g

SW,mg

WI,mg�g

72tg 8 3 22.68 210 9.2569wt 8 3 23.90 90 3.7674tg 8 3 23.98 270 11.2568wt 8 3 24.54 60 2.448tg 8 24 38.3 380 9.9224wt N�A 24 26.5 100 3.7750tg 10 3 21.7 200 9.2149wt 10 3 20.4 80 3.92148tg 8 6 26.97 240 8.89149wt 8 6 25.9 100 3.86156tg 10 6 23.44 280 11.94157wt 10 6 23.91 100 4.18220wt 8 7 23.13 120 5.2221tg 8 7 22.37 260 11.6222tg 8 7 21.67 250 11.5223wt 8 7 22.44 120 5.2

Mice were weighed after being killed, and the spleens were measured andweighed. Spleens of transgenic mice were enlarged, with a spleen weight�body-weight ratio three to four times greater than that of wild-type mice. BW,body weight; SW, spleen weight; WI, weight index.

Fig. 2. Transgenic mice, 6 months old, presented an enlarged abdomen andimportant splenomegaly. (A) Transgenic mice, 6 months old, had a consider-ably enlarged abdomen compared with wild-type mice, due to the clinicallyevident splenomegaly. (B) Spleens of the mice shown in A. The transgenicspleen is enlarged due to expansion of leukemic�lymphoma cells.

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spleens of transgenic mice at 3 weeks old (assessed on onetransgenic and one wild-type mouse) is 9% out of the entiregated lymphoid population and only 1.65% in the wild type; thisbecomes 6.6 � 1.4% in the spleen of 6-week-old transgenic mice(two transgenics analyzed from two different founding lines andtwo wild-type mice) and 4.7 � 0.3% for mice 7 weeks of age (twotransgenics and one wild type), while remaining unchanged forthe wild type. In the bone marrow of transgenic mice 6 monthsold, we found an increase of the pre-B cell population as definedby B220low�IgM� expression, compared with the wild type (Figs.5 and 6).

Forward-scatter analysis of the B220low�IgM� cell populationshowed that these cells are large blastoid cells (data not shown).

Cytogenetics Indicated Genomic Alterations. Cytogenetic studies ofthe karyotype of the splenocytes failed to identify consistentchromosomal abnormalities in the transgenic spleens comparedwith the normal littermates but showed occasionally somegenomic alterations (Fig. 7). These results indicate that theexpanded population of pre-B cells is diploid and cytogeneticallyquasinormal.

Southern Blot Analysis for Ig Heavy Chain Rearrangements Showed aPolyclonal Pre-B Cell Proliferation at Least Until 6 Weeks of Age.Southern blot analysis for clonality by V(D)J rearrangements onsplenocyte mouse DNA 3–6 weeks of age using multiple diges-tion enzymes did not show the presence of clonally rearrangedbands in the transgenic compared with wild-type mice (exceptfor one transgenic mouse that had a single prominant rearrangedband on all of the Southern blots performed with differentrestriction enzymes; data not shown). This shows that the B cellpopulation in mice of this age range was usually polyclonal,indicating that the lymphoproliferation was, for the most part,polyclonal, at least until 6 weeks of age (Fig. 8).

Microarray Analysis Confirmed the Up-Regulation of VpreB1 mRNA,Specific for the Pre-B Cells. Microarray analysis of miRNAs wasperformed on the total RNA extracted from the splenic whitecells of five transgenics (among which one did not have theexpression of the transgene) and six wild-type littermate coun-terparts, revealing a 10- to 20-fold increase in expression ofmiR155, -194, -224, -217, and -151 (Table 2, which is publishedas supporting information on the PNAS web site) and a decreaseof expression of miR146 and -138, 2- to 3-fold in transgenic miceoverexpressing miR155. Using an Affymetrix microarray chip,we studied the differential expression of mRNAs in the samegroup of transgenics, compared with their littermate controls.The statistical analysis of the Affymetrix microarray showed that200 proliferation genes were up-regulated, whereas 50 geneswere down-regulated in the miR155-overexpressing mice (datanot shown). Interestingly enough, the VpreB1 mRNA wasup-regulated, as one would expect when the proliferation ofpre-B cells takes place. These data complement the data fromflow cytometry analysis and immunohistochemistry (Table 3,which is published as supporting information on the PNAS website).

Fig. 4. IgM staining of the atypical lymphoid proliferation in the spleen of atransgenic mouse, 3 weeks old. IgM is present in the cytoplasm of the prolifer-ating lymphocytes (cIgM) as a brown perinuclear halo in the transgenic mice,whereas the wild-type lymphocytes are intensely brown, with no distinct nuclei,due to the presence of both sIgM and cIgM; immunohistochemistry (�400),spleen, mouse no. 50, 3 weeks old, malignant lymphoid cells with cIgM-positivestained cytoplasm.

Fig. 3. Histology and immunohistochemistry of transgenic spleens comparedwith the wild type. (a) H&E, spleen (�200), atypical lymphoid proliferationcompressing the white pulp in mouse no. 50 (founder 10), 3 weeks old. (b) H&E,spleen (�100), founder no. 8, 6 months of age. The overall architecture of thespleen is being replaced by the atypical lymphoid proliferation. There are only afew remaining germinal lymphoid follicles, greatly decreased in size, compressedby the proliferation. (c) H&E, spleen (�200), transgenic mouse, founder line no.8, 6 months old. The spleen architecture has been almost completely effaced bythe lymphoblastic proliferation. There are still visible remnants of two smallcompressed lymphoid follicules. (d) H&E, bone marrow (�400), transgenicmouse, founder line no. 8, 6 months old showing the lymphoblastic proliferationin the bone marrow that leads to the replacement of the hematopoietic foci. (e)H&E, normal spleen (�200). (f) Immunohistochemistry, spleen, transgenic mouseno. 72, 3 weeks old, for Ki67, showing increased lymphoid proliferation in thespleen (�200).

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E�-mmu-miR155 Transgenic Mice Present Pre-B Cell Proliferation andLymphoblastic Leukemia�High-Grade Lymphoma. We concluded,based on the flow cytometry, histological and immunohisto-chemical analyses, that a pre-B cell proliferation defined asIgM��CD43��TCR� occurred in the spleens and bone marrowsof transgenic mice, already detectable at 3 weeks of age. Thisproliferation led later on to splenomegaly, bone marrow replace-ment, and marked lymphopenia, features often associated withhigh-grade B cell malignancies. By the age of 6 months, seven ofseven transgenic mice developed high-grade B cell neoplasms(seven of seven transgenic mice, 6 months old) compared withthe wild-type controls, which were all healthy (11 of 11 wild-typemice). The transgenic mouse line not overexpressing miR155was also normal.

ConclusionAcute lymphoblastic leukemia and high-grade lymphoma arethe most common of leukemias and lymphomas in children.Transgenic mice with overexpression of miR155 develop alymphoproliferative disease resembling the human diseases,thus strongly suggesting that miR155 is directly implicated inthe initiation and�or progression of these diseases. The diseaseis, for the most part, polyclonal, suggesting that only theoverexpression of miR155 or additional few genetic changes is

sufficient to induce malignancy. Because malignancies, for themost part, are monoclonal, this finding suggests that miR155could be the downstream target of signal transduction path-ways activated in cancer. This is direct evidence that overex-pression of a miRNA results in the development of a neoplasticdisease, highlighting their potential role in human malignan-cies. Interestingly, we observed overexpression of miR155 insolid tumors such as breast, lung, and colon cancer (in lungcancers, overexpression of miR155 was an indicator of badprognosis; ref. 17). The E�-mmu-miR155 transgenic mousewill also be a useful tool to devise new therapeutic approachesto treat different forms of acute lymphoblastic leukemia orhigh-grade lymphomas in humans.

Materials and MethodsE�-mmu-miR155 Transgenic Mice. A 318-bp fragment was amplifiedby PCR from the 129SvJ mouse genome (The Jackson Laboratory)containing the precursor sequence of the miR155 and cloned intothe EcoRV and SalI sites of the pBSVE6BK (pE�) plasmid, whichhad been previously used by our group for the development ofchronic lymphocytic leukemia in E�-TCL1 transgenic mice (18),which contains the E� enhancer VH promoter for Ig heavy chains,alongside the 3� UTR and the poly(A) of the human �-globin gene(Fig. 1a) The transgene was isolated by cutting the construct withBssHII and PvuI and injected into the male pronucleus of fertilizedoocytes of pregnant FVB�N and C57BL�6 mice. Pups werescreened for the presence of the transgene by Southern blotperformed on tail-extracted DNA and digested with BamHI, usinga probe designed in the E� enhancer sequence. Fifteen founderswere identified (seven for C57BL�6, marked F1–7, and eight for theFVB�N strain, marked F8–15) and bred to age-matched wild-typemice (Fig. 1b). Transgenic hemizygous mice were born, studied, andcompared with their wild-type counterparts. Mice were genotypedby PCR performed on tail-extracted DNA (data not shown).

Northern Blot Analysis of Transgenic Offspring. Spleens were disso-ciated between two frosted slides, and the lysate was washed inPBS, depleted of red cells by hypotonic lysis with ammonium

Fig. 5. Flow cytometry analysis reveals an expansion of the B220low�CD10low�IgM��CD5��TCR��CD43� population in the spleen and bone marrow oftransgenic mice. Flow cytometry analysis of the spleen and bone marrow wasused to characterize the immunophenotypic profile of the lymphocytes in thespleen (a) and bone marrow (b) of mice coming from two different lines offounders (founders 8 and 10), with ages between 3 weeks and 6 months. (a)Gated splenocytes for two transgenic mice and two wild types, 3 weeks of age(Tg no. 74, F8; and WT no. 68, F10) and 7 weeks of age (Tg no. 156; and WT no.157). The upper left quadrant gating the B220��IgM� population shows anincrease of the precursor B cells, in comparison with wild type. (b) Gated bonemarrow white cells for one transgenic and one wild-type mouse, 6 months ofage (Tg no. 8 and WT no. 24). The upper right quadrant indicates the decreaseof the B200� IgM� gated mature B cell population of the bone marrow.

Fig. 6. CD10 expression evaluated by flow cytometry on B220� gatedsplenocytes in transgenic and wild-type mice. Flow cytometry analysis onB220� gated splenocytes of transgenic and wild-type mice, 7 weeks of age,showing an increase in the transgenic compared with the wild type. The rightcolumn shows the B220� gated population in transgenic and wild-type mice.The increase of the B220low population is noticeable (intercalated betweenthe two peaks of B220� and B220�) in the transgenic mouse. The left columnshows the increase in percentage of the CD10� population in the B220� gatedpopulation only, in transgenics and wild-type mice, proving that the B220low

proliferation is due, at least in part, to an increase of the CD10� population.

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chloride (NH4Cl), centrifuged, and resuspended in PBS. TotalRNA was extracted with TRIzol (GIBCO, Invitrogen), loadedand denatured on SDS�PAGE, and blotted on a Hybond N�

membrane (Amersham Pharmacia). The membrane was hybrid-ized with a �-32P radioactive probe represented by the antisenseof the mature mmu-miR155 sequence, incubated overnight,washed, and exposed to a PhosphorImager screen (MolecularDynamics). The image was processed by using a Typhoon imageprocessing system (Amersham Biosciences) (Fig. 1c).

Somatic Measurements. Mice were weighed after being killed, andthe spleens were measured and weighed.

WBC and Smear Preparation. Blood was drawn from retroorbitalblood vessels, smeared on frosted slides, and Giemsa-stained;

part of it was centrifuged, washed in PBS, and treated withammonium chloride, then cells were counted with a cell-counterchamber.

Flow Cytometry Analysis. Single-cell suspension of splenocytesand bone marrow cells was depleted of mature red blood cellsby hypotonic lysis (0.165 M NH4Cl) and stained with thefollowing conjugated antibodies: anti-B220-phycoerythrin,anti-IgM-FITC, anti-TCR-phycoerythrin cy5, anti-CD5-phycoerythrin, and anti-CD-43-FITC (all antibodies werefrom BD PharMingen). Flow cytometry was carried out on aBecton Dickinson FACSCalibur, and data were analyzed byusing the Becton Dickinson FACS CONVERT 1.0 for Macsoftware.

Fig. 7. Chromosome 9 abnormality, identified by a thick extra band. Cytogenetics of the lymphoid cells isolated from a transgenic spleen. Splenocytes weregrown and assessed for chromosomal deletions, translocations, inversions, and number of metaphases. Few abnormalities were identified, but none seemed tobe consistently present in all of the samples analyzed.

Fig. 8. Ig heavy chain rearrangement. Southern blot on DNA extracted from the splenocytes of transgenic and wild-type mice. Southern blot on transgenicand wild-type DNA extracted from splenocytes (five transgenic and four wild-type mice, 3–6 weeks of age) using the JH4 probe and different digesting enzymes:StuI, BglII, BamHI, and HindIII. The thick bands of high molecular weight correspond to the germ line; there are no rearranged bands in the transgenics, comparedwith the wild type (TG, transgenic mice; WT, wild-type mice).

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Histology and Immunohistochemistry. Mice were necropsied, andspleens, femurs, and sternums were fixed in 10% bufferedformalin, included in paraffin, and then cut at 4 �m. Sectionswere stained with H&E according to standard protocols. For thedewaxing step, sections were heated for 1 h at 55°C, followed byrehydration steps through a graded ethanol series and distilledwater, immersed in PBS, and then treated with 0.1% trypsinsolution in Tris buffer for 30 min at 37°C. Endogenous peroxi-dase was blocked with 10% normal serum. CD43, B220, andVpreB1 (CD179a) were used as primary antimouse antibody(BD PharMingen). Secondary antibodies and diaminobenzidinewere added according to the manufacturer’s instruction.

Ig Heavy Chain Gene Rearrangements. A probe was designed andamplified in the JH4 fragment of the Ig heavy chain region onthe mouse genomic, using the following primers: forward, 5�-TGAAGGATCTGCCAGAACTGAA-3�, and reverse, 5�-TGCAATGCTCAGAAAACTCCAT-3�.

Spleens of the transgenic and wild-type mice were dissociatedbetween frosted slides in PBS, treated with ammonium chloridefor the erythrocyte lysis, centrifuged, and resuspended in PBS.DNA was extracted from the spleen white cells and digested withEcoRI, StuI, BglII, BamHI, and HindIII. Digested DNA wasblotted on a Hybond N� membrane, hybridized with the JH4probe radioactively labeled with 32P, then exposed to a Phos-phorImager screen, and processed by using a Typhoon scanner.

Microarray Analysis. mRNA and miRNA gene transcriptional profiling.Total RNA isolation was performed with the TRIzol method(Invitrogen), according to the manufacturer’s instructions.miRNA expression profiling. RNA labeling and hybridization onmiRNA microarray chips were performed as described (19).Briefly, 5 �g of total RNA from each sample was biotin-labeledby reverse transcription using 5� biotin end-labeled randomoctomer oligo primer. Hybridization of biotin-labeled cDNA wascarried out on a miRNA microarray chip (Ohio State University,Ver. 2.0), which contains 800 miRNA probes, including 245human and 200 mouse miRNA genes, in quadruplicate. Hybrid-ization signals were detected by biotin binding of a streptavidin–Alexa647 conjugate b using Axon Scanner 4000B (Axon Instru-

ments, Union City, CA). The images were quantified by GENEPIX6.0 software (Axon Instruments).mRNA expression profiling. GeneChip Mouse genome 430 2.0 arrays(Affymetrix), containing probe sets for �45,000 characterizedgenes and expressed sequence tags, were used. Sample labelingand processing, GeneChip hybridization, and scanning wereperformed according to Affymetrix protocols. Briefly, double-stranded cDNA was synthesized from total RNA with theSuperScript Choice System (Invitrogen), with a T7 RNA poly-merase promoter site added to its 3� end (Genset, La Jolla, CA).Biotinylated cRNAs were generated from cDNAs in vitro andamplified by using the BioArray T7 RNA polymerase labeling kit(Enzo Diagnostics). After purification of cRNAs by the RNeasymini kit (Qiagen, Hilden, Germany), 20 �g of cRNA wasfragmented at 94°C for 35 min. Approximately 12.5 �g offragmented cRNA was used in a 250-�l hybridization mixturecontaining herring-sperm DNA (0.1 mg�ml; Promega), plusbacterial and phage cRNA controls (1.5 pM BioB, 5 pM BioC,25 pM BioD, and 100 pM Cre) to serve as internal controls forhybridization efficiency. Aliquots (200 �l) of the mixture werehybridized to arrays for 18 h at 45°C in a GeneChip HybridizationOven 640 (Affymetrix). Each array was washed and stained withstreptavidin–phycoerythrin (Invitrogen) and amplified with bio-tinylated anti-streptavidin antibody (Vector Laboratories) onthe GeneChip Fluidics Station 450 (Affymetrix). Arrays werescanned with the GeneArray G7 scanner (Affymetrix) to obtainimage and signal intensities.

Cytogenetics. Femur bone marrow was flushed with RPMI me-dium 1640�20% FBS and collected into 5 ml of RPMI medium1640�20% FBS with heparin 1%. Cells were grown and assessedfor chromosomal deletions, translocations, inversions, and num-ber of metaphases.

We thank Xin-An Pu for technical help with the creation of thetransgenic mouse and Nicole White, Bryan McElwain, and RickMeissner for technical assistance with the f low cytometry analysis.This study was supported by a National Cancer Institute grant (toC.M.C.).

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