EμmiR-155转基因小鼠前B细胞的增值和淋巴母细胞白血病、高分级淋巴瘤的研究

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,400West 12th Avenue,Columbus,OH 43210Contributed by Carlo M.Croce,March 22,2006MicroRNAs (miRNAs)represent a newly discovered class of post-transcriptional regulatory noncoding small RNAs that bind to targeted mRNAs and either block their translation or initiate their degradation.miRNA profiling of hematopoietic lineages in humans and mice showed that some miRNAs are differentially expressed during hematopoietic development,suggesting a role in hemato-poietic cell differentiation.In addition,recent studies suggest the involvement of miRNAs in the initiation and progression of cancer.miR155and BIC,its host gene,have been reported to accumulate in human B cell lymphomas,especially in diffuse large B cell lymphomas,Hodgkin lymphomas,and certain types of Burkitt lymphomas.Here,we show that E ␮-mmu-miR155transgenic mice exhibit initially a preleukemic pre-B cell proliferation evident in spleen and bone marrow,followed by frank B cell malignancy.These findings indicate that the role of miR155is to induce polyclonal expansion,favoring the capture of secondary genetic changes for full transformation.transgenic mouse ͉malignant lymphoproliferation ͉microRNAsMicroRNAs (miRNAs)represent a new class of abundant small 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 1993in Caenorhabditis elegans (1),there have been numerous reports that implicated these tiny molecules in the posttranscriptional regulation of a large array of proteins with very diverse roles,ranging from cell prolifera-tion and differentiation to lipid metabolism (2–6).miRNA profiling of hematopoietic lineages in humans and mice showed that miRNAs are differentially expressed in the course of hematopoietic development,suggesting a potential role in hematopoietic differentiation (7–9).We have shown that miR-15a and miR-16-1are deleted or down-regulated in Ϸ68%of cases of chronic lymphocytic leukemia (CLL)(10,11),and that miRNAs genes are frequently located at fragile sites and other genomic regions involved in cancers (12).Transcripts of miR155and BIC (its host gene)transcripts have been shown to accumulate in human B cell lymphomas,especially diffuse large B cell lymphomas (13),Hodgkin lymphomas (14),and subsets of Burkitt lymphomas (latency type III Epstein–Barr virus-positive Burkitt lymphoma;ref.15).These reports provide indirect evidence that miR155may play a role in B cell development and lymphomagenesis.We have also reported that miR155is 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 B cell malignancy.Results and DiscussionProduction and Characterization of E ␮-mmu-miR155.We generatedtransgenic mice in which the expression of mmu-miR155(mousemiR155)is under the control of a V H promoter-Ig heavy chain E ␮enhancer,which becomes active at the late pro-B cell stage of the B cell development.Fifteen transgenic founders were identified by Southern blot hybridization,seven on C57BL ͞B6and eight on FVB ͞N backgrounds.These were bred to wild-type mice of the same strain to produce 15independent transgenic lines.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 expressed the transgene.Wild-type mice did not express mature miR155in the spleen,as reported previously (16).E ␮-mmu-miR155Exhibited Splenomegaly as Early as 3Weeks of Age.Spleens of transgenic mice were enlarged,with a spleen weight ͞body weight ratio three to four times greater than that of wild-type mice at 3weeks of age and did not increase signifi-cantly with age (Table 1and Fig 2).Transgenic Mice Overexpressing miR155Are Leukopenic.The whiteblood cell count (WBC)of transgenics 3months of age was 10ϫ106Ϯ1ϫ106͞ml compared with 40ϫ106Ϯ1.5ϫ106͞ml for normal age-matched mice.The WBC for transgenic mice 6months of age was even lower,at 6ϫ106Ϯ0.5ϫ106͞ml,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 multiple foci of secondary hematopoiesis (Fig.3a ).Mice at 6months of age presented histologically a greatly increased malignant lym-phoid population with marked atypia and blastic appearance,proliferating in the vascular channels of the red pulp and gradually replacing the white pulp.The number of lymphoid follicles was decreased,and the overall architecture of the spleen was distorted by lymphoid proliferation (Fig.3b and c ).A histologically similar lymphoid population was present in the bone marrow of 6-month-old mice.Expression of the prolifer-ation Ki67antigen showed a marked lymphoid proliferation in transgenic mice,not observed in wild-type mice (Fig.3f ).Immunohistochemistry performed to identify the immuno-phenotype of the lymphoid proliferation showed low positivity of the atypical expanded lymphocytes for B220and VpreB1(CD179a),although CD43was negative (data not shown).The IgM staining of the paraffin-embedded sections of the trans-genic spleens showed the presence of ␮chains in the cytoplasmConflict 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@.©2006by The National Academy of Sciences of the USA7024–7029͉PNAS ͉May 2,2006͉vol.103͉no.18 ͞cgi ͞doi ͞10.1073͞pnas.0602266103of the proliferating lymphocytes (Fig.4),whereas flow cytom-etry analysis failed to identify the expression of IgM on the surface of these cells,indicating that the expanded lymphoid cells expressed cytoplasmic ␮chain but did not express surface IgM.Flow Cytometry Analysis of E ␮-mmu-miR155Reveals an Expansion of the B220low ͞CD10low ͞IgM ؊͞CD5؊͞TCR ؊͞CD43؊Population.Flow cy-tometry analysis,performed on single-cell suspensions of WBC from spleens and bone marrows of transgenic and wild-type mice of 3,6,and 7weeks and 6months of age,showed an increase of B220low ͞CD10low ͞IgM Ϫ͞CD5Ϫ͞TCR Ϫ͞CD43Ϫlymphoid cells in 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 in human acute lymphoblastic leukemia or lymphoblastic lym-phoma.Our findings indicate that the B220low ͞CD19low ͞CD10low ͞IgM Ϫ͞TCR Ϫ͞CD43Ϫlymphoid population intheFig.1.Production and characterization of E ␮-mmu-miR155.(a )A construct for the miR155transgene was designed as shown.The mmu-miR155was cloned between the EcoRV and SalI sites,putting the transgene under the control of the V H promoter E ␮enhancer.The construct was then injected in the male pronuclei of the oocytes of pregnant C57BL ͞6and FVB ͞N female mice.(b )Southern blot was used to genotype the founders.Fifteen transgenic founders were born,seven on a C57BL ͞6background (b Left ,lanes 1,3,5,6,7,10,and 14are transgenics,and lanes 2,4,8,9,11,12,13,and 15are wild types)and eight on an FVB ͞N background (b Right ,lanes 1,3,5,7,9,11,13,and 15are transgenics,and lanes 2,4,6,8,10,12,and 14are wild types).These were then bred to wild-type strain-matched mice to produce 15transgenic lines.(c )In each of the 15transgenic lines,expression of the transgene was assessed by Northern blot on total RNA extracted from the lymphocytes isolated from the spleens of 3-week-old mice by using the antisense oligonucleotide of the mmu-miR155mature sequence as a probe.Five of the transgenic lines with the highest level of expression of the mature miR155in 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 transgenic compared with wild-type miceMice Line (founder)Age,wks BW,g SW,mg WI,mg ͞g 72tg 8322.682109.2569wt 8323.9090 3.7674tg 8323.9827011.2568wt 8324.5460 2.448tg 82438.33809.9224wt N ͞A 2426.5100 3.7750tg 10321.72009.2149wt 10320.480 3.92148tg 8626.972408.89149wt 8625.9100 3.86156tg 10623.4428011.94157wt 10623.91100 4.18220wt 8723.13120 5.2221tg 8722.3726011.6222tg 8721.6725011.5223wt8722.441205.2Mice were weighed after being killed,and the spleens were measured and weighed.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,weightindex.Fig.2.Transgenic mice,6months old,presented an enlarged abdomen and important splenomegaly.(A )Transgenic mice,6months old,had a consider-ably enlarged abdomen compared with wild-type mice,due to the clinically evident splenomegaly.(B )Spleens of the mice shown in A .The transgenic spleen is enlarged due to expansion of leukemic ͞lymphoma cells.Costinean et al.PNAS ͉May 2,2006͉vol.103͉no.18͉7025M E D I C A L S C I E N C ESspleens of transgenic mice at 3weeks old (assessed on one transgenic and one wild-type mouse)is 9%out of the entire gated lymphoid population and only 1.65%in the wild type;this becomes 6.6Ϯ1.4%in the spleen of 6-week-old transgenic mice (two transgenics analyzed from two different founding lines and two wild-type mice)and 4.7Ϯ0.3%for mice 7weeks of age (two transgenics and one wild type),while remaining unchanged for the wild type.In the bone marrow of transgenic mice 6months old,we found an increase of the pre-B cell population as defined by B220low ͞IgM Ϫexpression,compared with the wild type (Figs.5and 6).Forward-scatter analysis of the B220low ͞IgM Ϫcell population showed that these cells are large blastoid cells (data not shown).Cytogenetics Indicated Genomic Alterations.Cytogenetic studies of the karyotype of the splenocytes failed to identify consistent chromosomal abnormalities in the transgenic spleens compared with the normal littermates but showed occasionally some genomic alterations (Fig.7).These results indicate that the expanded population of pre-B cells is diploid and cytogenetically quasinormal.Southern Blot Analysis for Ig Heavy Chain Rearrangements Showed a Polyclonal Pre-B Cell Proliferation at Least Until 6Weeks of Age.Southern blot analysis for clonality by V(D)J rearrangements on splenocyte mouse DNA 3–6weeks of age using multiple diges-tion enzymes did not show the presence of clonally rearranged bands in the transgenic compared with wild-type mice (except for one transgenic mouse that had a single prominant rearranged band on all of the Southern blots performed with different restriction enzymes;data not shown).This shows that the B cell population in mice of this age range was usually polyclonal,indicating that the lymphoproliferation was,for the most part,polyclonal,at least until 6weeks of age (Fig.8).Microarray Analysis Confirmed the Up-Regulation of VpreB1mRNA,Specific for the Pre-B Cells.Microarray analysis of miRNAs wasperformed on the total RNA extracted from the splenic white cells of five transgenics (among which one did not have the expression of the transgene)and six wild-type littermate coun-terparts,revealing a 10-to 20-fold increase in expression of miR155,-194,-224,-217,and -151(Table 2,which is published as supporting information on the PNAS web site)and a decrease of expression of miR146and -138,2-to 3-fold in transgenic mice overexpressing ing an Affymetrix microarray chip,we studied the differential expression of mRNAs in the same group of transgenics,compared with their littermate controls.The statistical analysis of the Affymetrix microarray showed that 200proliferation genes were up-regulated,whereas 50genes were down-regulated in the miR155-overexpressing mice (data not shown).Interestingly enough,the VpreB1mRNA was up-regulated,as one would expect when the proliferation of pre-B cells takes place.These data complement the data from flow 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 a transgenic mouse,3weeks 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 (ϫ400),spleen,mouse no.50,3weeks old,malignant lymphoid cells with cIgM-positive stainedcytoplasm.Fig.3.Histology and immunohistochemistry of transgenic spleens compared with the wild type.(a )H&E,spleen (ϫ200),atypical lymphoid proliferation compressing the white pulp in mouse no.50(founder 10),3weeks old.(b )H&E,spleen (ϫ100),founder no.8,6months of age.The overall architecture of the spleen is being replaced by the atypical lymphoid proliferation.There are only a few remaining germinal lymphoid follicles,greatly decreased in size,compressed by the proliferation.(c )H&E,spleen (ϫ200),transgenic mouse,founder line no.8,6months old.The spleen architecture has been almost completely effaced by the lymphoblastic proliferation.There are still visible remnants of two small compressed lymphoid follicules.(d )H&E,bone marrow (ϫ400),transgenic mouse,founder line no.8,6months old showing the lymphoblastic proliferation in the bone marrow that leads to the replacement of the hematopoietic foci.(e )H&E,normal spleen (ϫ200).(f )Immunohistochemistry,spleen,transgenic mouse no.72,3weeks old,for Ki67,showing increased lymphoid proliferation in the spleen (ϫ200).7026͉ ͞cgi ͞doi ͞10.1073͞pnas.0602266103Costinean etal.E ␮-mmu-miR155Transgenic Mice Present Pre-B Cell Proliferation and Lymphoblastic Leukemia ͞High-Grade Lymphoma.We concluded,based on the flow cytometry,histological and immunohisto-chemical analyses,that a pre-B cell proliferation defined as IgM Ϫ͞CD43Ϫ͞TCR Ϫoccurred in the spleens and bone marrows of transgenic mice,already detectable at 3weeks of age.This proliferation led later on to splenomegaly,bone marrow replace-ment,and marked lymphopenia,features often associated with high-grade B cell malignancies.By the age of 6months,seven of seven transgenic mice developed high-grade B cell neoplasms (seven of seven transgenic mice,6months old)compared with the wild-type controls,which were all healthy (11of 11wild-type mice).The transgenic mouse line not overexpressing miR155was also normal.ConclusionAcute lymphoblastic leukemia and high-grade lymphoma are the most common of leukemias and lymphomas in children.Transgenic mice with overexpression of miR155develop a lymphoproliferative disease resembling the human diseases,thus strongly suggesting that miR155is directly implicated in the initiation and ͞or progression of these diseases.The disease is,for the most part,polyclonal,suggesting that only the overexpression of miR155or additional few genetic changes issufficient to induce malignancy.Because malignancies,for the most 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 neoplastic disease,highlighting their potential role in human malignan-cies.Interestingly,we observed overexpression of miR155in solid tumors such as breast,lung,and colon cancer (in lung cancers,overexpression of miR155was an indicator of bad prognosis;ref.17).The E ␮-mmu-miR155transgenic mouse will also be a useful tool to devise new therapeutic approaches to treat different forms of acute lymphoblastic leukemia or high-grade lymphomas in humans.Materials and MethodsE ␮-mmu-miR155Transgenic Mice.A 318-bp fragment was amplifiedby PCR from the 129SvJ mouse genome (The Jackson Laboratory)containing the precursor sequence of the miR155and cloned into the EcoRV and SalI sites of the pBSVE6BK (pE ␮)plasmid,which had been previously used by our group for the development of chronic lymphocytic leukemia in E ␮-TCL1transgenic mice (18),which contains the E ␮enhancer V H promoter for Ig heavy chains,alongside the 3ЈUTR and the poly(A)of the human ␤-globin gene (Fig.1a )The transgene was isolated by cutting the construct with BssHII and PvuI and injected into the male pronucleus of fertilized oocytes of pregnant FVB ͞N and C57BL ͞6mice.Pups were screened for the presence of the transgene by Southern blot performed on tail-extracted DNA and digested with BamHI,using a probe designed in the E ␮enhancer sequence.Fifteen founders were identified (seven for C57BL ͞6,marked F1–7,and eight for the FVB ͞N strain,marked F8–15)and bred to age-matched wild-type mice (Fig.1b ).Transgenic hemizygous mice were born,studied,and compared with their wild-type counterparts.Mice were genotyped by 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 in PBS,depleted of red cells by hypotonic lysis withammoniumFig.5.Flow cytometry analysis reveals an expansion of the B220low ͞CD10low ͞IgM Ϫ͞CD5Ϫ͞TCR Ϫ͞CD43Ϫpopulation in the spleen and bone marrow of transgenic mice.Flow cytometry analysis of the spleen and bone marrow was used to characterize the immunophenotypic profile of the lymphocytes in the spleen (a )and bone marrow (b )of mice coming from two different lines of founders (founders 8and 10),with ages between 3weeks and 6months.(a )Gated splenocytes for two transgenic mice and two wild types,3weeks of age (Tg no.74,F 8;and WT no.68,F 10)and 7weeks of age (Tg no.156;and WT no.157).The upper left quadrant gating the B220ϩ͞IgM Ϫpopulation shows an increase of the precursor B cells,in comparison with wild type.(b )Gated bone marrow white cells for one transgenic and one wild-type mouse,6months of age (Tg no.8and WT no.24).The upper right quadrant indicates the decrease of the B200ϩIgM ϩgated mature B cell population of the bonemarrow.Fig.6.CD10expression evaluated by flow cytometry on B220ϩgated splenocytes in transgenic and wild-type mice.Flow cytometry analysis on B220ϩgated splenocytes of transgenic and wild-type mice,7weeks of age,showing an increase in the transgenic compared with the wild type.The right column shows the B220ϩgated population in transgenic and wild-type mice.The increase of the B220low population is noticeable (intercalated between the two peaks of B220Ϫand B220ϩ)in the transgenic mouse.The left column shows the increase in percentage of the CD10ϩpopulation in the B220ϩgated population only,in transgenics and wild-type mice,proving that the B220low proliferation is due,at least in part,to an increase of the CD10ϩpopulation.Costinean et al.PNAS ͉May 2,2006͉vol.103͉no.18͉7027M E D I C A L S C I E N C ESchloride (NH 4Cl),centrifuged,and resuspended in PBS.Total RNA was extracted with TRIzol (GIBCO,Invitrogen),loaded and denatured on SDS ͞PAGE,and blotted on a Hybond N ϩmembrane (Amersham Pharmacia).The membrane was hybrid-ized with a ␥-32P radioactive probe represented by the antisense of the mature mmu-miR155sequence,incubated overnight,washed,and exposed to a PhosphorImager screen (Molecular Dynamics).The image was processed by using a Typhoon image processing 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 retroorbital blood vessels,smeared on frosted slides,and Giemsa-stained;part of it was centrifuged,washed in PBS,and treated with ammonium chloride,then cells were counted with a cell-counter chamber.Flow Cytometry Analysis.Single-cell suspension of splenocytesand bone marrow cells was depleted of mature red blood cells by hypotonic lysis (0.165M NH 4Cl)and stained with the following conjugated antibodies:anti-B220-phycoerythrin,anti-IgM-FITC,anti-TCR-phycoerythrin cy5,anti-CD5-phycoerythrin,and anti-CD-43-FITC (all antibodies were from BD PharMingen).Flow cytometry was carried out on a Becton Dickinson FACSCalibur,and data were analyzed by using the Becton Dickinson FACS CONVERT 1.0for Macsoftware.Fig.7.Chromosome 9abnormality,identified by a thick extra band.Cytogenetics of the lymphoid cells isolated from a transgenic spleen.Splenocytes were grown and assessed for chromosomal deletions,translocations,inversions,and number of metaphases.Few abnormalities were identified,but none seemed to be consistently present in all of the samplesanalyzed.Fig.8.Ig heavy chain rearrangement.Southern blot on DNA extracted from the splenocytes of transgenic and wild-type mice.Southern blot on transgenic and wild-type DNA extracted from splenocytes (five transgenic and four wild-type mice,3–6weeks of age)using the JH4probe 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,compared with the wild type (TG,transgenic mice;WT,wild-type mice).7028͉ ͞cgi ͞doi ͞10.1073͞pnas.0602266103Costinean etal.Histology and Immunohistochemistry.Mice were necropsied,and spleens,femurs,and sternums were fixed in10%buffered formalin,included in paraffin,and then cut at4␮m.Sections were stained with H&E according to standard protocols.For the dewaxing step,sections were heated for1h at55°C,followed by rehydration steps through a graded ethanol series and distilled water,immersed in PBS,and then treated with0.1%trypsin solution in Tris buffer for30min at37°C.Endogenous peroxi-dase was blocked with10%normal serum.CD43,B220,and VpreB1(CD179a)were used as primary antimouse antibody (BD PharMingen).Secondary antibodies and diaminobenzidine were added according to the manufacturer’s instruction.Ig Heavy Chain Gene Rearrangements.A probe was designed and amplified in the JH4fragment of the Ig heavy chain region on the mouse genomic,using the following primers:forward,5Ј-TGAAGGATCTGCCAGAACTGAA-3Ј,and reverse,5Ј-TGCAATGCTCAGAAAACTCCAT-3Ј.Spleens of the transgenic and wild-type mice were dissociated between frosted slides in PBS,treated with ammonium chloride for the erythrocyte lysis,centrifuged,and resuspended in PBS. DNA was extracted from the spleen white cells and digested with EcoRI,StuI,BglII,BamHI,and HindIII.Digested DNA was blotted on a Hybond Nϩmembrane,hybridized with the JH4 probe radioactively labeled with32P,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 on miRNA microarray chips were performed as described(19). Briefly,5␮g of total RNA from each sample was biotin-labeled by reverse transcription using5Јbiotin end-labeled random octomer oligo primer.Hybridization of biotin-labeled cDNA was carried out on a miRNA microarray chip(Ohio State University, Ver.2.0),which contains800miRNA probes,including245 human and200mouse miRNA genes,in quadruplicate.Hybrid-ization signals were detected by biotin binding of a streptavidin–Alexa647conjugate b using Axon Scanner4000B(Axon Instru-ments,Union City,CA).The images were quantified by GENEPIX 6.0software(Axon Instruments).mRNA expression profiling.GeneChip Mouse genome4302.0arrays (Affymetrix),containing probe sets forϾ45,000characterized genes and expressed sequence tags,were used.Sample labeling and processing,GeneChip hybridization,and scanning were performed according to Affymetrix protocols.Briefly,double-stranded cDNA was synthesized from total RNA with the SuperScript Choice System(Invitrogen),with a T7RNA poly-merase promoter site added to its3Јend(Genset,La Jolla,CA). Biotinylated cRNAs were generated from cDNAs in vitro and amplified by using the BioArray T7RNA polymerase labeling kit (Enzo Diagnostics).After purification of cRNAs by the RNeasy mini kit(Qiagen,Hilden,Germany),20␮g of cRNA was fragmented at94°C for35min.Approximately12.5␮g of fragmented cRNA was used in a250-␮l hybridization mixture containing herring-sperm DNA(0.1mg͞ml;Promega),plus bacterial and phage cRNA controls(1.5pM BioB,5pM BioC, 25pM BioD,and100pM Cre)to serve as internal controls for hybridization efficiency.Aliquots(200␮l)of the mixture were hybridized to arrays for18h at45°C in a GeneChip Hybridization Oven640(Affymetrix).Each array was washed and stained with streptavidin–phycoerythrin(Invitrogen)and amplified with bio-tinylated anti-streptavidin antibody(Vector Laboratories)on the GeneChip Fluidics Station450(Affymetrix).Arrays were scanned with the GeneArray G7scanner(Affymetrix)to obtain image and signal intensities.Cytogenetics.Femur bone marrow was flushed with RPMI me-dium1640͞20%FBS and collected into5ml of RPMI medium 1640͞20%FBS with heparin1%.Cells were grown and assessed for chromosomal deletions,translocations,inversions,and num-ber of metaphases.We thank Xin-An Pu for technical help with the creation of the transgenic mouse and Nicole White,Bryan McElwain,and Rick Meissner for technical assistance with the flow cytometry analysis. 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