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Transgenic Human 5 Rescues the Murine 5 Nullizygous Phenotype¹

Mary E. Donohoe^2,*, Gabriele B. Beck-Engeser, Nils Lonberg, Hajime Karasuyama^§, Richard L. Riley^*, Hans-Martin Jäck^,¶ and Bonnie B. Blomberg^3,*

^* Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101; Department of Microbiology and Immunology, Loyola University, Stritch School of Medicine, Maywood, IL 60153; GenPharm International, San Jose, CA 95131; ^§ Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; and ^¶ Division of Molecular Immunology, Department of Internal Medicine IV, University of Erlangen, Erlangen, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The human 5 (hu5) gene is the structural homologue of the murine 5 (m5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The 5 and VpreB polypeptides together with the Ig µ H chain and the signal-transducing subunits, Ig and Igß, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu5 gene. High levels of expression of the transgenic hu5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu5 polypeptide on the cell surface and in association with mouse Ig µ and mouse VpreB. Finally, we found that the hu5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m5^-/- background. Therefore, we conclude that the hu5 polypeptide can biochemically and functionally substitute for m5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
B lymphocyte development is distinguished by the ordered rearrangement of the Ig H chain (HC)⁴ and L chain (LC) gene segments and the temporal expression of distinct B lineage-restricted genes such as those encoding the surrogate LC (SLC), 5, and VpreB (1, 2). The mouse SLC genes, m5 and mVpreB, were initially identified by their exclusive expression in pre-B cells (3, 4, 5, 6). The SLC together with a rearranged Ig µ (µ) HC and the Ig-associated transducing chains, Ig and Igß, comprise the pre-B cell receptor (pre-BCR) (7, 8, 9). The exact function of the SLC, including 5, is unknown, but the pre-BCR apparently plays a critical role in B lineage differentiation as targeted disruption of the m5 gene blocks B lymphocyte development at the transition between the pro-B cell and large pre-B cell stages and drastically reduces the number of mature B lymphocytes in the periphery (10, 11, 12). It has been hypothesized that the pre-BCR provides a signal for B lineage differentiation from the pro-B to pre-B cell stages (10, 13, 14, 15, 16, 17) and allelic exclusion at the early or large pre-B cell stage (18, 19). Pre-B cell signaling has also been implicated for cell survival/proliferation (12) and to screen for functional µ chains (20, 21) and thus influence the V_H repertoire (20, 21, 22, 23). Alternatively, it has been proposed that the SLC may deliver the pre-BCR-expressing cell to the appropriate cellular compartment for subsequent activation (24). Whether a pre-BCR ligand exists has still not been established, but recent evidence suggests that at least the proliferative and differentiative functions of the pre-BCR may be stromal independent and hence inferred, ligand independent (25). The precise nature of the pre-BCR differentiation signal has not been identified, but recently a mitogen-activated protein kinase has been implicated following Igß cross-linking in pro-B cells (26).

The mouse genes for the SLC are expressed exclusively at the pro-B and pre-B cell stage and may serve as markers of differentiation. This selective pro-B/pre-B cell expression of the m5 and mVpreB genes is controlled at the transcriptional level (27, 28, 29) and likely involves pro-B/pre-B cell-specific transactivating factors or B-specific repressing factors acting in conjunction with cis-elements in the SLC promoters and enhancers. Recently, a locus control region (LCR) was described for the m5/mVpreB locus (30). The m5 promoter initiates transcription at multiple start sites and contains two distinct regulatory regions, an upstream regulatory region that confers tissue and stage specificity and a basal promoter element that confers gene transcription in multiple cell lineages albeit at a much lower level in a T cell line (31, 32, 33). The transcription factors EBF (early B cell factor) and E47 are important for the pre-B cell-specific expression of m5 (34, 35).

A human homologue to the m5 gene has been referred to as the lambda-like, 14.1, or pseudo-light chain (36, 37, 38). The human homologue to mVpreB is referred to as huVpreB (39). Chang et al. referred to the human homologue to m5 as 14.1 due to its location on an EcoRI genomic fragment of 14 kb (36). In this paper, we will refer to the 14.1 gene as hu5 (human 5). The expression of a functional hu5 gene is crucial for B lymphopoiesis as a patient with a null mutation of the hu5 gene has a severe B cell deficit and aggamaglobulinema due to a complete block at the pro-B to pre-B cell stage (40). Similar to the m5 gene, the hu5 gene is expressed at the pro-B and pre-B cell stages (16, 37, 41, 42, 43, 44, 45). To begin to identify potential cis-regulatory elements important for the early B lineage expression of the hu5 gene, our laboratory analyzed the hu5 gene locus for sites of nucleosome reconfigurations using DNase I hypersensitivity mapping (45). DNase I hypersensitive sites (HS) have been found to be associated with promoters, enhancers, LCRs, and matrix attachment regions (46, 47). Two pre-B cell-specific HS, localized 2.4 kb upstream and at the start of the first exon of the hu5 gene, were identified (45).

As an additional experimental approach to study the stage- and tissue-specific expression of the hu5 gene in vivo, we generated hu5 transgenic mice containing a 28-kb genomic fragment encompassing the hu5 gene. This is the first report to describe a human SLC transgenic mouse. In this study, we demonstrate that the hu5 transgene confers a high level of hu5 expression at the mRNA and protein levels that is restricted to the pro-B and pre-B cell stages within the B cell lineage. Thus, the hu5 transgene contains the necessary cis-regulatory elements for pro-B/pre-B lymphocyte expression. These hu5 transgenic mice were also used to ask whether the hu5 protein could assemble with mVpreB and mµHC to form a chimeric human/mouse pre-BCR and rescue the block in B lineage development in m5-deficient mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of hu5 transgenic mice

Hu5 transgenic mice were generated by pronuclear microinjection of a gel-purified 28-kb XhoI fragment from the hu5-containing cosmid clone, Hu18 (45, 48) (Fig. 1) into half-day mouse embryos (49). Three independent transgenic mouse lines were generated, each contained 10 copies of the hu5-encompassing 28-kb XhoI fragment.

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Restriction map of the hu5-containing cosmid clone, Hu18, used to generate the hu5 transgenic mice. The XhoI gel-purified fragment was used for microinjection, and the 3.6-kb BamHI hu5-specific probe was used to screen mouse tail biopsies for the presence of the hu5 transgene. The three exons of the hu5 gene are depicted by the boxes. The downward arrows map the location of the DNase I HS. The downward triangle marks the location of the CpG island (45 48 ). Restriction enzymes: B, BamHI; Bg, BglI; Bs, BssHII; E, EagI; H, HindIII; Hc, HincII; K, KpnI; M, MluI; Nh, NheI; Nr, NruI; P, PstI; R, EcoRI; S, SstI; S2, SstII; Sm, SmaI; X, XhoI; Xb, XbaI.

Identification of mice containing hu5 genomic sequences

The presence of hu5 transgene sequences was detected by Southern blot hybridization of biopsied tail DNA. Genomic DNA was isolated from tail biopsies by proteinase K digestion followed by phenol-chloroform extraction and isopropanol precipitation (49). Hu5 sequences were detected in BamHI-digested genomic DNA by Southern blot analysis with a specific ³²P-labeled 3.6-kb BamHI fragment derived from Hu18 (Fig. 1). The copy number of the hu5 transgenic mice was determined by comparing the hu5 hybridization signal of the human cell line Jurkat with the biopsied tail genomic DNA. The amount of hybridization signals on films was estimated densitometrically by scanning with the IS-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA) and normalized to the quantity of DNA loaded. Prehybridization and hybridization was at 42°C in 50% formamide as previously described (45).

The m5 nullizygous, m5^-/- (10), and their wild-type, m5^+/+, control mice (strain 129) were generously provided by Drs. John Kearney (University of Alabama, Birmingham, AL) and Werner Müller (University of Cologne, Cologne, Germany) with permission of Drs. Fritz Melchers (Basel Institute of Immunology, Basel, Switzerland) and Klaus Rajewsky (University of Cologne). Neo, m5, and hu5 probes were used to confirm the genotypes of the m5^-/- (10), hu5^+/-, and hu5^+/-m5^-/- mice by analyses of tail biopsies. The 2.0-kb SalI bacterial neomycin resistance gene (neo) probe (derived from pCMV.Neo; Ref. 50) was used to identify the presence of the neo gene within the targeted disruption of the m5 gene (10). Homozygous deletions of the m5 gene, m5^-/- as above, mice were characterized by having mutant-sized 2.5-kb hybridizing neo bands in a EcoRI digest of double intensity. The m5^-/- genotype was also confirmed by hybridization to a 0.8-kb BglII m5 probe (31) similar to the method of Kitamura et al. (10) and/or by specific genomic DNA PCR for m5, neo, and hu5. Because the three lines of hu5 transgenic mice had approximately the same copy number (10) of hu5, one of the homozygous hu5 mouse lines was crossed to m5 nullizygous mice. The 16-kb EcoRI-hybridizing hu5 band identified mice with at least one copy of the hu5 transgene. Progeny were genotyped as above and the m5^-/-, hu5^+/-m5^-/-, hu5^+/-m5^+/-, and hu5^+/-m5^+/+ mice were selected for further analyses.

Preparation of Abelson murine leukemia viral (A-MuLV)-transformed pre-B cell lines and other permanent cell lines

A-MuLV-transformed cell lines were prepared from bone marrow cells from three hu5^+/-m5^-/- mice from one transgenic line. Approximately 3 x 10⁷ cells were resuspended in 24 ml RPMI 1640 media with 10% FCS, of which 20 ml was filtered supernatant containing Abelson virus and 24 µl of polybrene (hexadimethrine bromide) added to a final concentration of 8 µg/ml. The Abelson virus supernatant was harvested directly from growing 54CL4 cells (51). Cells were incubated with access to 5% CO₂ at 37° for 2 h then centrifuged and resuspended in media including 5 x 10^-5 M 2-ME with 10% FCS. Liquid cultures were set up in 24-well plates at 2 x 10⁵ cells/well. Cell growth was visible after about 10 days, and cells were subsequently subcloned to generate µ-producing or µ-nonproducing lines.

Permanent (transformed) human and mouse cell lines used for fluorescent Ab staining, RT-PCR, and/or immunoprecipitation were as follows. Human lines, 697 and Nalm-6, are acute lymphoblastic leukemias (52, 53), representative of pre-B cells, expressing hu5, huVpreB, and huµ proteins, previously described (43, 45). Abelson lines (A-MuLV) expressing m5, mVpreB, and mµ proteins were Tµ, previously described (20), and 107.2.

Fluorescent flow cytometric cell analysis of ex vivo cells and permanent cell lines containing hu5 genomic sequences

Single-cell suspensions from bone marrow were prepared by flushing cells from femur and tibia pairs with PBS. Spleen cell suspensions were prepared by lysing RBC with a hypotonic ammonium chloride buffer (ACK). Cell-surface Ags were measured by two- or three-color immunofluorescence analyses. Half a million cells were stained with FITC-, PE-, or biotin-conjugated Abs in FACS buffer (HBSS, buffered in 0.02% sodium azide and 0.1% BSA) for 30 min on ice, protocol as previously described (54). Cells were stained directly with FITC-conjugated goat anti-mouse IgM (Boehringer Mannheim, Indianapolis, IN) and PE-conjugated anti-CD43 (mAb S7; PharMingen, San Diego, CA). Cells were also stained indirectly with biotin-conjugated anti-B220 (anti-CD45 mAb 6B2; PharMingen) followed by streptavidin-Cychrome (PharMingen). The anti-hu5 (HSL-11) mAb was generated as published (55) and was detected by FITC-conjugated rat anti-mouse 1 Abs. Fluorescence was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) for 5,000–10,000 cells.

For cell sorting, bone marrow and spleen cells were stained directly with FITC-conjugated goat anti-mouse IgM (Boehringer Mannheim) and indirectly stained with biotinylated anti-B220 (anti-CD45 mAb 6B2; PharMingen) followed by streptavidin-Cychrome (PharMingen, San Diego, CA). Bone marrow cells were sorted to separate pro-B/pre-B cell (B220⁺IgM^-) fractions from B cell (B220⁺IgM⁺) fractions. Spleen cells were sorted to separate B cell (B220⁺IgM⁺) fractions from non-B cell (B220^-IgM^-) fractions. Sorted cell populations were reanalyzed and showed >98% purity. Cell sorting was performed on a FACStar^Plus sorter (Becton Dickinson).

Flow cytometric analysis of the m5^-/-hu5^+/-, hu5^+/-, and m5^-/- mice were performed as described above using three-color staining (as described by Hardy et al. (56)). Cells were directly stained with FITC-conjugated anti-B220 (mAb 6B2; Caltag, South San Francisco, CA), PE-conjugated anti-CD43 (PharMingen), and PE-conjugated anti-IgM (PharMingen).

Hu5 transgene expression determined by RT-PCR analysis

RNA was isolated from various mouse tissues or cell lines using the TRIzol reagent (Life Technologies, Gaithersburg, MD). Two micrograms of total RNA was reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Foster City, CA). The transgene-encoded cDNA sequences were amplified using the GeneAmp PCR kit with AmpliTaq DNA polymerase following manufacturer’s instruction (Perkin-Elmer). All reverse transcriptase (RT) reactions and PCR were performed in a tissue culture hood using nonaerosal tips (Fisher Scientific, Pittsburgh, PA). The PCR amplification was performed using a Thermolyne Amplitron thermocycler (Thermolyne, Dubuque, IA). The gene-specific primers used for PCR are: hu5 forward, 5'-CGCCCAACAGCTGCATCGCA-3', hu5 reverse, 5'-GGCCAGTCCAGGAGCCGCGC-3'; m5 forward, 5'-ATGAAGCTCAGAGTAGGACA-3', m5 reverse, 5'-TCTTTAAGGAAGGCAGGAAC-3'; GAPDH forward, 5'-ACCACAGTCCATGCCATCAC-3', GAPDH reverse, 5'-TCCACCACCCTGTTGCTGTA-3'.

The hu5 gene-specific forward oligonucleotide primer corresponds to the sequence 117 bp downstream of the translational start for the hu5 gene within exon 1 and the hu5 primers are given above. PCR amplification using these hu5 gene-specific primers gave a PCR product of 122 bp. The m5-specific primers are given above, which correspond to the sequences located at the initial methionine of exon 1 and amino acid number 114 within exon 3, respectively (5). PCR amplification using these m5-specific primers yields a 361-bp PCR product. The murine GAPDH gene was detected using gene-specific primers initially obtained from Clontech Laboratories (Palo Alto, CA). The GAPDH primers are given above, and PCR yields a 454-bp PCR product. PCR amplified products were electrophoresed in 1.5% or 2% agarose gels, transferred to nitrocellulose membrane, and subjected to hybridization with the following probes.

To detect the hu5 PCR product, a hu5-specific exon I, 0.18-kb BglI, probe was used (44). A m5-specific 0.8-kb PstI-KpnI probe that encompasses the first exon of m5 was used to detect m5 and a 452-bp PCR-generated GAPDH probe was used to detect the GAPDH PCR product. Poly(A)⁺ mRNA was extracted from sorted bone marrow and spleen cell populations using the Quick Prep Micro mRNA purification kit following manufacturer’s instructions (Pharmacia Biotech, Piscataway, NJ). The extracted mRNA was resuspended in 10 µl dimethyl pyrocarbonate-treated distilled water. Two microliters of mRNA were used for each RT reaction as described above. PCR primers and PCR amplification conditions of cDNA are described below for hu5 expression in particular cell stages of the sorted populations. RT-PCR signals were quantitated using a Molecular Dynamics Phosphoimager SF model 455 (Sunnyvale, CA).

Immunoprecipitation analysis

Immunoprecipitations were done essentially as previously described (20). Briefly, 5 x 10⁶ cells/ml were labeled metabolically with 75 µCi/ml Trans ³⁵S label (1076 Ci/mmol; ICN Pharmaceuticals, Costa Mesa, CA) for 3 h and lysed on ice for 30 min in NaCl/EDTA/Tris (NET) lysis buffer. Proteins were precipitated from lysates with mAbs against mouse µ (rat IgG anti-mouse Cµ2 Ab, b7-6), human µ (goat anti-hu IgM, Southern Biotechnology Association, Birmingham, AL), anti-m5 (FS-1) (57), or anti-hu5 (HSL-11) (55). The anti-m5 Ab, FS-1, was generously provided by Dr. Jan Jongstra (57). Secondary Abs (mouse IgG anti-rat IgG; Pierce, Rockford, IL, or goat anti-mouse for hu5) and Staphylococcus aureus were used for immunoprecipitation. Immunoprecipitated proteins were separated by Laemmli SDS-PAGE on 12.5% gels and detected by fluorography.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of mice expressing the hu5 transgene

To generate transgenic hu5 mice, we injected mouse pronuclei with a 28-kb XhoI genomic fragment encompassing all three exons of the hu5 gene, the hu5 promoter region, the two introns, 9 kb upstream of the first exon, and 12 kb downstream of the third exon. This 28-kb hu5 genomic fragment (Fig. 1) contains the two pre-B lymphocyte-specific DNase I hypersensitive sites (45).

Using Southern blot analysis with a hu5-specific probe, a 3.6-kb BamHI fragment (Fig. 1), three hu5 founders were identified each with an average of 10 transgene copies (data not shown). Offspring were screened for the presence of the transgenic hu5 and endogenous m5 gene segments by Southern blot and PCR analysis as described in Materials and Methods.

The hu5 transgene is expressed in the bone marrow, thymus, and testis

To detect the expression of the hu5 transgene at the RNA level, total RNA was analyzed from various tissues of transgenic and nontransgenic littermates by RT-PCR using hu5 gene-specific forward and reverse primers. These hu5 gene-specific oligonucleotides were synthesized to specifically amplify hu5 sequences, but not the endogenous m5 or mIg genes. A similar strategy was designed to detect m5 RNA. Primers specific for the housekeeping gene GAPDH were used to control for the integrity of the RNA preparation and for semiquantitative estimates of relative RNA levels.

The above RT-PCR strategy was applied to total RNA prepared from various tissues of adult male and female hu5 transgenic mice. We found that hu5 expression in a heterozygous female mouse was limited to the bone marrow and thymus (data not shown). In a heterozygous transgenic male mouse, the hu5 transgene was expressed in the bone marrow, thymus, and testis (Fig. 2A, upper panel). Expression of the m5 gene in a hu5 transgene mouse was, as expected (3, 5, 6, 26, 27), predominantly in the bone marrow and was not detected in the testis of a male transgenic mouse (Fig. 2A, middle panel). Very low levels of m5 RNA were detected in the thymus (see Discussion). Therefore, the presence of transgenic hu5 transcripts did not alter the tissue-specific expression of the m5 gene.

View larger version (75K): [in this window] [in a new window]   FIGURE 2. Tissue distribution of hu5 and m5 mRNA in hu5 transgenic mice. A, RT-PCR was performed on total RNA prepared from several hu5+/- female and hu5+/- male mice from various tissues (bone marrow, thymus, testis, heart, liver, kidney, brain, ovary, lung, and bone marrow) and representative data shown. The PCR products were subjected to Southern blot analysis and hybridized to hu5-, GAPDH-, and m5-specific probes. Positive controls for hu5 and m5 were prepared from RNA using the cell lines 697 and 107.2, respectively. B, Stage distribution of hu5 and m5 mRNA in hu5 transgenic mice. RT-PCR was performed on poly(A)+ mRNA prepared from bone marrow cells sorted for pro-B/pre-B (B220+ sIgM-) and B cell (B220+ sIgM+) populations as indicated in Materials and Methods. Dilutions of cDNA are indicated above each lane. PCR products were subjected to Southern blot analysis and hybridized to the probes in A. mRNA prepared from 697 and 107.2 were used as positive controls for hu5 and m5 expression, respectively. Data shown in this panel were from films exposed for 17.5 h for hu5 (including 697), 2 h for m5 (including 107.2), and 0.5 h for GAPDH. Calculations for relative hu5 and m5 expression and for pro B/pre B vs B expression were done with consistent 0.5 h exposures and analyzed on a phosphoimager. Hu5 and m5 mRNA are predominately expressed in the bone marrow pro-pre-B cell populations. Splenic B cell (B220+IgM+) and non-B cell (B220-IgM-) populations were also sorted, and neither hu5 nor m5 were expressed in these populations (data not shown).

hu5

hu5

hu5

hu5

Hu5 expression is limited to the pro-B/pre-B lymphocyte fraction within the B lineage

To determine whether the hu5 transgene is expressed in a stage-specific fashion within the B lymphocyte lineage, pro-B/pre-B (B220⁺, surface (s)IgM^-) and B cells (B220⁺, sIgM⁺) were sorted by FACS analysis from the bone marrow of an hu5 transgenic (m5^+/+) mouse. RT-PCR was performed with poly(A)⁺-mRNA from the above B lymphoid fractions of a hu5 transgenic mouse, and PCR products were analyzed on Southern blots using the 0.18-kb BglI hu5-specific probe. The transgenic hu5 gene is predominately expressed in the pro-B/pre-B populations (B220⁺, sIgM^-) in the bone marrow (Fig. 2B). In addition, expression of the m5 gene was restricted to the same B lymphoid cell population (Fig. 2B). After normalizing the hu5 and m5 signals to the GAPDH signals and analyzing similar (30 min) exposures on the phosphoimager, the pro-B/pre-B population (B220⁺, sIgM^-) expresses 5.9–8.8 times more hu5 (at 1:16 and 1:4 dilutions, respectively) and about 10.2 times more m5 (at the 1:4 dilution) mRNA transcripts as does the B cell population (B220⁺, sIgM⁺). This indicates that expression of hu5, as has been previously shown for m5, (3, 4, 5, 27, 28) is largely restricted to the pro-B/pre-B cell populations. These results are consistent with the hu5 transgene being expressed in a stage-specific fashion within the B cell lineage. To compare hu5 and m5 expression within the pro-B/pre-B population (after multiplying the hu5 signal by three to take into account its smaller fragment size), m5 is 2.8–3.4 times higher than hu5 expression (1:16 and 1:4 dilutions, respectively).

The hu5 protein is expressed in a stage-specific fashion in hu5 transgenic mice

To determine whether the hu5 transgene is expressed at the protein level, we membrane-stained ex vivo bone marrow cells from transgenic hu5^+/- and nontransgenic m5^+/+ wild-type littermates with fluorochrome-conjugated anti-CD43 and anti-B220 Abs (Fig. 3, A and E). The cells were fixed, permeablilized, and stained for hu5 with the monoclonal mouse IgG1 anti-human 5 Ab, HSL11 (55), followed by the FITC-conjugated rat anti-mouse 1 Ab. The data presented in Fig. 3 reveal that CD43⁺ B220^low pro-B/early pre-B cells (Fig. 3B), as well as the CD43^- B220^low pre-B/immature B cells (Fig. 3C) from transgenic hu5 mice stain positively, whereas pro-B/early pre-B cells from nontransgenic m5 ^+/+ wild-type littermates did not react with the anti-hu5 Ab (Fig. 3F). In addition, as expected from the hu5 transgenic RNA expression studies (Fig. 2), CD43^- B220^high mature B cells from hu5 transgenic bone marrow also fail to react with the monoclonal anti-hu5 Ab (Fig. 3D). Attempts to detect hu5 on the surface of ex vivo bone marrow cells failed as has been reported by others for m5 (58). We conclude from these data that the hu5 transgene is expressed at the RNA and protein levels in a stage-specific fashion.

View larger version (52K): [in this window] [in a new window]   FIGURE 3. Flow cytometric analysis of hu5 protein expression in ex vivo hu5 transgenic mice bone marrow cells. Bone marrow cells from a hu5+/- transgene on a wild-type m5+/+ background (A–D) or wild-type m5+/+ (E and F) mice were stained for membrane CD43 and B220. Cells were fixed, permeablilized, and cytoplasmically stained for hu5 with HSL11 and rat anti-mouse 1-FITC. A, Membrane CD43 and B220 staining on bone marrow cells prepared from a hu5+/-m5+/+ mouse. The percentage of pro-B/early pre-B, (R2), pre-B/immature-B (R3), and mature B (R4) are shown. B, Percentage of hu5 protein expressed cytoplasmically in the gated pro-B/early pre-B (R2) bone marrow cell subpopulations from A. C, Percentage of hu5 protein expressed cytoplasmically in gated pre-B/immature-B cells (R3). D, Mature B bone marrow (subpopulation R4 from A) cells do not react with the anti-hu5 Ab. E, Membrane CD43 and B220 staining on bone marrow cells prepared from a wild-type (m5+/+) mouse. Gated B lineage populations are depicted in the boxes as in A. F, Hu5 cytoplasmic staining of the wild-type mouse pro-B/early pre-B cell population (R2 of E).

The hu5 transgene rescues the m5 null phenotype

One line of hu5 transgenic mice was crossed with m5 nullizygous m5^-/- mice to investigate whether the hu5 transgene could rescue the null phenotype. Offspring were analyzed for the presence of the hu5 transgene and the genotype at the m5 locus as described in Materials and Methods. Flow cytometric analyses performed on bone marrow cells from transgenic heterozygous hu5 (hu5^+/-m5^+/+) mice show normal numbers of CD43⁺/B220⁺ pro-B (4.8%), CD43^-/B220⁺ pre-B/immature B cells (9.9%), and CD43^-/B220⁺ B cells (3.8%) (Fig. 4A). In contrast, the nontransgenic m5^-/- nullizygous mice have elevated pro-B (9.0%), very low CD43^low/B220⁺ pre-B/immature B cells (2.1%), and, as described previously (10), virtually no B cells (0.2%) (Fig. 4B). In hu5 transgenic mice with homozygous deletions at the m5 gene, m5^-/-, bone marrow cells have increased numbers of CD43^-/B220⁺ pre-B/immature B cells (9.9%) and CD43^-/B220⁺ B cells (5.8%) (Fig. 4C) similar to that observed in hu5^+/-m5^+/+ mice. These results have been reproduced in >10 independent experiments (data not shown) and with mice from a variety of ages. Similar results were obtained when we performed a three-color flow cytometric analysis with Abs against CD43, B220, and the pre-B cell marker CD25 (data not shown). Cellularity in all mice was roughly identical. Therefore, we conclude that the hu5 transgene is able to reconstitute the block at the CD43⁺/B220⁺ stage in the m5^-/- mice.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. Flow cytometric analysis of B lineage markers on bone marrow cells from 8-wk-old hu5+/-m5+/+ (A), m5-/- (B), and hu5+/-m5-/- mice (C). Expression of cell-surface Ags was measured by two-color immunofluorescence analyses. This figure shows a representative experiment using Abs to CD43 and B220 (as in Fig. 3). Percentages of B lineage populations are shown as in depicted in Fig. 3, A and E.

Abelson cell lines from hu5 transgenic mice produce a hu5 protein that is expressed on the cell surface and binds mVpreB and µHC

Because the hu5 transgene rescued the block at the CD43⁺/B220⁺ stage in m5 nullizygous mice, we expected to detect mouse µ and hu5 chains on the surface of A-MuLV-transformed pre-B cell lines prepared from hu5^+/-m5^-/- mice. A priori, we expected to detect hu5 polypeptide in association with mVpreB and mµ as a chimeric mouse/human pre-BCR in A-MuLV-transformed pre-B cell lines prepared from these mice. To address these predictions, we generated A-MuLV-transformed pre-B cell lines from bone marrow cells of transgenic heterozygous hu5^+/-m5^-/- mice. The cell lines, which we named Hula (for human lambda 5 Abelson lines), were screened for synthesis of cytoplasmic µ with fluorochrome-conjugated anti-µ Abs. Because we (21) and others (23) found that most murine µ chains using the VH81X gene segment fail to pair with m5 and are therefore not transported to the cell surface, we eliminated cells that stained with an anti-VH81X-specific anti-idiotypic antiserum (L. Hartwell et al., manuscript in preparation). Hula cultures containing µ-positive cells were subcloned by limiting dilution or sorted for surface mµ, and single-cell clones positive for mµ were saved for further analysis. In addition, to rule out the possibility that surface µ-positive cells were due to Dµ proteins reaching the surface in the absence of hu5, the presence of a full-length (VDJ)µ rearrangement was determined by Southern analysis with D and JH probes on Hula genomic DNA (data not shown).

A total of five independent cytoplasmic µ-positive Hula clones were screened for surface expression of µ and analyzed for hu5 by flow cytometric analysis. The independent clones showed unique V(D)J rearrangement patterns. A representative experiment is shown for Hula9 in Fig. 5. Hula9 (H9) shows cell-surface expression of membrane mµ (Fig. 5A) at levels comparable to that of the A-MuLV-transformed pre-B cell lines 107.2 (Fig. 5B) or TK.µ (data not shown) (20). H9 does express membrane hu5 (Fig. 5C), whereas the control (wild-type) 107.2 cell line does not (<1%) (Fig. 5D). As expected, Abs against m5 react only with 107.2 cells (Fig. 5F) but not with Hula cells (Fig. 5E). Because others have shown that mµ chains that fail to pair with 5 chains are not transported to the cell surface (20, 22) and full-length mµ requires m5 and mVpreB to reach the cell surface and for differentiation past the early pre-B cell stage (58), we conclude that these results indicate that the hu5 protein is able to carry the mµ and most likely mVpreB to the cell surface.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Membrane fluorescent staining of hu5+/-m5-/- Hula lines. A-MuLV-transformed cell lines were prepared from bone marrow cells from the hu5+/-m5-/- mice. Hula line H9 (38A1 (5 ) H9) was analyzed for synthesis of membrane mµ (A), hu5 (C), m5 (E), and murine (G) and compared with the mouse Abelson line, 107.2, known to be positive for mµ, m5, and . The Hula H9 cells are positive for membrane staining of mµ (A) and hu5 (C), but not for (G). The 107.2 cells are positive for mµ (B), m5 (F), and m (H), but negative for staining with the anti-hu5 (HSL11) Ab (D).

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View larger version (73K): [in this window] [in a new window]   FIGURE 6. Analysis of immunoprecipitated mouse and human pre-BCR in A-MuLV-transformed cells lines prepared from hu5+/-m5-/- mice (Hula cell lines). Mouse and human pre-BCR (µ, 5, VpreB) were analyzed in 35S-labeled cellular extracts from Hula lines, the µ-positive mouse A-MuLV pre-B cell line, Tµ, and the human pre-B acute lymphoblastic leukemia line, Nalm-6, in anti-µ and anti-hu5 precipitates. A, Anti-mµ precipitation of hu5 and mVpreB in Nalm-6 (lane 1), Hula lines H9 and H11 (lanes 2 and 3), and Tµ (lane 4). Secondary (2°) Ab (mouse anti-rat IgG) alone showed no specific bands precipitated (lanes 5–7). B, Anti-hu5 precipitation of cellular extracts from Hula subclones H11 (lane 1), H10 (lane 2), H9 (lanes 3 and 7), and 45.1 (lane 6) show coprecipitation of mVpreB and mµ. The control Nalm-6 (lanes 4 and 8) cell line coprecipitates huVpreB and hu µ. The mouse A-MuLV pre-B cell line, Tµ (lane 5), was used as a negative control for the anti-hu5 Ab. Data from two different hu5+/-m5-/- mice (H for H9, H10, H11; 45 for 45.1) are shown.

	Discussion

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These results suggest that the 28-kb hu5 genomic fragment contains all the necessary cis-regulatory elements for pro-B/pre-B cell expression in an in vivo mouse model. We did observe mRNA expression of hu5 in the bone marrow as well as thymus and testis in three independent lines of hu5 transgenic mice. These mice did express hu5 message, as well as a very small amount of m5 message in the thymus, suggesting that a common stem cell precursor and/or low levels of pre-B cells may be present in the mouse thymus. The relative increased expression of hu5 mRNA as compared with m5 in the thymus may be attributable to the copy number of the hu5 transgene, although quantitation of hu5 and m5 expression in pro B/pre B cells (Fig. 2B) indicates that hu5 is not overexpressed in its natural pre-B cell compartment. Hu5 protein was not detected in the thymus nor was there an alteration in the normal relative ratios of CD4/CD8 T cells in the thymus of a transgenic vs a nontransgenic littermate. This may likely be due to the absence of VpreB and µ, which may decrease stability of the 5 polypeptide.

Our data also reveal that the hu5 message is detected at a relatively high level in the testis of transgenic mice. Other transgenes have been shown to be deregulated or abnormally expressed in germ tissue (60). The testis does not contain histones (61), therefore histone deacetylation as a possible transcriptional repression mechanism within the 28-kb hu5 genomic fragment may be missing in this tissue. Although it is possible that a negative regulatory region is missing resulting in testicular (and thymus) expression, we also cannot rule out the possible effects of the site of chromosome integration.

The organization of the SLC gene loci are different between humans and mice. In the human, a single VpreB gene has been reported, whereas at least two exist in the mouse (38). Three -like genes have been described in humans, one of which is functional, (14.1 or hu5). A single functional m5 exists in the mouse, as indicated by mice nullizygous for the 5 gene, which have a block at the transition between the pro-B/pre-B cell stage; however, this defect is leaky as older mice are able to recover 20% of their B cells by 4 mo of age. In contrast to the human locus, in which the hu5 gene is approximately a megabase distal to the VpreB locus (48), the m5 locus is situated 4.5 kb 3' to the VpreB1 gene (6). A number of pre-B cell-specific HS within the m5 locus have been mapped (32), and this region has been identified as an LCR capable of regulating both VpreB and 5 (30). The genomic regions for the hu5 locus have not been fully characterized, although HS 1 (Fig. 1A) may be the site of a cis-regulatory region. The fact that the hu5 transgene showed high RNA expression in the thymus as compared with m5 may be due to the transgene missing a negative regulatory element for thymus. As all three lines had a similar expression, it is less likely that the site of integration selectively allowed for thymic expression.

The results presented in this report show that the hu5 transgene produces a protein that is able to function in place of the m5 protein as determined by the pre-B/immature B and B cell numbers in the bone marrow of m5^-/- mice. Association of mµ with the m5 and mVpreB proteins is necessary to get cell-surface expression of mµ (8, 20, 58). Recently Minegishi et al. (62) demonstrated that the unique 50-aa region of the hu5 protein, located carboxyl-terminal to the signal peptide, serves as an intramolecular chaperone to prevent folding of hu5 protein in the absence of its partner, VpreB. Without this unique region, the hu5 protein can be secreted in the absence of VpreB. Our results suggest that the unique 50-aa region of hu5 (which shows the highest percent amino acid difference from the mouse at 50%) is not hindering mVpreB or mµ association. The presence of mVpreB is likely a crucial player in assembly of the mouse/human chimeric pre-BCR in hu5 transgenic mice with deletions of both m5 alleles, by extension from the mouse studies (8, 20, 22, 63). Consistent with the Minegishi et al. report (62), our results suggest that the unique amino-terminal region of the hu5 protein can function in the m5^-/- mouse for assembly into the chimeric pre-BCR. Our results do show a weaker or less stable association of the hu5/mVpreB/mµ complex, which may reflect differences in the hu5 sequence.

Differences in early B cell development exist between humans and mice. The IL-7 receptor plays an important role in early B lymphocyte signaling in mouse but not in human B cell development (64, 65). In contrast, in human, but not mouse B cell development, mutations in the Bruton’s tyrosine kinase (Btk) gene result in a B cell block at the pro-B/pre-B cell transition (66). At least for the pre-BCR, our results suggest that enough conservation of 5 exists between mouse and human to allow for structural and functional similarities.

We have created an in vivo mouse model for the hu5 gene. Results presented in this paper indicate that most of the necessary cis-regulatory regions are contained in a 28-kb XhoI genomic fragment encompassing the hu5 gene. We show that the hu5 protein can assemble with mµ and mVpreB to form a chimeric pre-BCR that rescues the m5^-/- phenotype. These mice will provide a valuable reagent for understanding the molecular requirements for a functional mouse and human pre-BCR.

	Acknowledgments

 
We thank Drs. Werner Muller, Klaus Rajewsky, Fritz Melchers, and John Kearney for the m5 nullizygous mice and control strain 129 mice. We thank Dr. Barbara A. Malynn at Center for Blood Research, Harvard Medical School for critical comments on the paper. We thank Jim Phillips for flow cytometric analysis assistance, we appreciate excellent technical assistance from Marta Perez, Karen Kamm, Alim Ladha, Andrea Young, Krischan Hudson, and Stephanie Beasley, and we appreciate excellent secretarial support from Pat Washington and Michelle Perez.

	Footnotes

 
¹ This work was supported by an R03 Grant, AG14892, and funds from the Sylvester Comprehensive Cancer Center and from the University of Miami School of Medicine (to B.B.B.), Grant AG 15474 (to R.L.R.), and a Junior Faculty Research Award from the American Cancer Society of America, grants from the American Cancer Society (Illinois Division), the Tobacco Research Council of America, and the National Cancer Institute (R29) (to H.-M.J.).

² Current address: Harvard Medical School, Department of Pathology, 200 Longwood Avenue, Boston, MA 02115.

³ Address correspondence and reprint requests to Dr. Bonnie B. Blomberg, University of Miami School of Medicine, Department of Microbiology and Immunology, P.O. Box 016960 (R-138), Miami, FL 33101.

⁴ Abbreviations used in this paper: HC, H chain; LC, L chain; hu5, human 5; HS, hypersensitive; LCR, locus control region; m5, mouse 5; SLC, surrogate light chain; µ, Ig µ; BCR, B cell receptor; A-MuLV, Abelson murine leukemia virus; RT, reverse transcriptase; s, surface; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Received for publication July 13, 1999. Accepted for publication March 1, 2000.

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