Transgenic Human λ5 Rescues the Murine λ5 Nullizygous Phenotype

Generation of huλ5 transgenic mice

Huλ5 transgenic mice were generated by pronuclear microinjection of a gel-purified 28-kb XhoI fragment from the huλ5-containing cosmid clone, Huλ18 (45, 48) (Fig. 1⇓) into half-day mouse embryos (49). Three independent transgenic mouse lines were generated, each contained ∼10 copies of the huλ5-encompassing 28-kb XhoI fragment.

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FIGURE 1.

Restriction map of the huλ5-containing cosmid clone, Huλ18, used to generate the huλ5 transgenic mice. The XhoI gel-purified fragment was used for microinjection, and the 3.6-kb BamHI huλ5-specific probe was used to screen mouse tail biopsies for the presence of the huλ5 transgene. The three exons of the huλ5 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 huλ5 genomic sequences

The presence of huλ5 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). Huλ5 sequences were detected in BamHI-digested genomic DNA by Southern blot analysis with a specific ³²P-labeled 3.6-kb BamHI fragment derived from Huλ18 (Fig. 1⇑). The copy number of the huλ5 transgenic mice was determined by comparing the huλ5 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 mλ5 nullizygous, mλ5^−/− (10), and their wild-type, mλ5^+/+, 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, mλ5, and huλ5 probes were used to confirm the genotypes of the mλ5^−/− (10), huλ5^+/−, and huλ5^+/−mλ5^−/− 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 mλ5 gene (10). Homozygous deletions of the mλ5 gene, mλ5^−/− as above, mice were characterized by having mutant-sized 2.5-kb hybridizing neo bands in a EcoRI digest of double intensity. The mλ5^−/− genotype was also confirmed by hybridization to a 0.8-kb BglII mλ5 probe (31) similar to the method of Kitamura et al. (10) and/or by specific genomic DNA PCR for mλ5, neo, and huλ5. Because the three lines of huλ5 transgenic mice had approximately the same copy number (10) of huλ5, one of the homozygous huλ5 mouse lines was crossed to mλ5 nullizygous mice. The 16-kb EcoRI-hybridizing huλ5 band identified mice with at least one copy of the huλ5 transgene. Progeny were genotyped as above and the mλ5^−/−, huλ5^+/−mλ5^−/−, huλ5^+/−mλ5^+/−, and huλ5^+/−mλ5^+/+ 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 huλ5^+/−mλ5^−/− mice from one transgenic line. Approximately 3 × 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 × 10⁻⁵ M 2-ME with 10% FCS. Liquid cultures were set up in 24-well plates at 2 × 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 huλ5, huVpreB, and huμ proteins, previously described (43, 45). Abelson lines (A-MuLV) expressing mλ5, mVpreB, and mμ proteins were Tκμ, previously described (20), and 107.2.

Fluorescent flow cytometric cell analysis of ex vivo cells and permanent cell lines containing huλ5 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-huλ5 (HSL-11) mAb was generated as published (55) and was detected by FITC-conjugated rat anti-mouse γ1 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 mλ5^−/−huλ5^+/−, huλ5^+/−, and mλ5^−/− 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).

Huλ5 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: huλ5 forward, 5′-CGCCCAACAGCTGCATCGCA-3′, huλ5 reverse, 5′-GGCCAGTCCAGGAGCCGCGC-3′; mλ5 forward, 5′-ATGAAGCTCAGAGTAGGACA-3′, mλ5 reverse, 5′-TCTTTAAGGAAGGCAGGAAC-3′; GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′, GAPDH reverse, 5′-TCCACCACCCTGTTGCTGTA-3′.

The huλ5 gene-specific forward oligonucleotide primer corresponds to the sequence 117 bp downstream of the translational start for the huλ5 gene within exon 1 and the huλ5 primers are given above. PCR amplification using these huλ5 gene-specific primers gave a PCR product of 122 bp. The mλ5-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 mλ5-specific primers yields a 361-bp PCR product. The murine GAPDH gene was detected using γ 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 huλ5 PCR product, a huλ5-specific exon I, 0.18-kb BglI, probe was used (44). A mλ5-specific 0.8-kb PstI-KpnI probe that encompasses the first exon of mλ5 was used to detect mλ5 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 huλ5 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 × 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-mλ5 (FS-1) (57), or anti-huλ5 (HSL-11) (55). The anti-mλ5 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 κ for huλ5) and Staphylococcus aureus were used for immunoprecipitation. Immunoprecipitated proteins were separated by Laemmli SDS-PAGE on 12.5% gels and detected by fluorography.

Generation of mice expressing the huλ5 transgene

To generate transgenic huλ5 mice, we injected mouse pronuclei with a 28-kb XhoI genomic fragment encompassing all three exons of the huλ5 gene, the huλ5 promoter region, the two introns, 9 kb upstream of the first exon, and 12 kb downstream of the third exon. This 28-kb huλ5 genomic fragment (Fig. 1⇑) contains the two pre-B lymphocyte-specific DNase I hypersensitive sites (45).

Using Southern blot analysis with a huλ5-specific probe, a 3.6-kb BamHI fragment (Fig. 1⇑), three huλ5 founders were identified each with an average of 10 transgene copies (data not shown). Offspring were screened for the presence of the transgenic huλ5 and endogenous mλ5 gene segments by Southern blot and PCR analysis as described in Materials and Methods.

The huλ5 transgene is expressed in the bone marrow, thymus, and testis

To detect the expression of the huλ5 transgene at the RNA level, total RNA was analyzed from various tissues of transgenic and nontransgenic littermates by RT-PCR using huλ5 gene-specific forward and reverse primers. These huλ5 gene-specific oligonucleotides were synthesized to specifically amplify huλ5 sequences, but not the endogenous mλ5 or mλIg genes. A similar strategy was designed to detect mλ5 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 huλ5 transgenic mice. We found that huλ5 expression in a heterozygous female mouse was limited to the bone marrow and thymus (data not shown). In a heterozygous transgenic male mouse, the huλ5 transgene was expressed in the bone marrow, thymus, and testis (Fig. 2⇓A, upper panel). Expression of the mλ5 gene in a huλ5 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. 2⇓A, middle panel). Very low levels of mλ5 RNA were detected in the thymus (see Discussion). Therefore, the presence of transgenic huλ5 transcripts did not alter the tissue-specific expression of the mλ5 gene.

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FIGURE 2.

Tissue distribution of huλ5 and mλ5 mRNA in huλ5 transgenic mice. A, RT-PCR was performed on total RNA prepared from several huλ5^+/− female and huλ5^+/− 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 huλ5-, GAPDH-, and mλ5-specific probes. Positive controls for huλ5 and mλ5 were prepared from RNA using the cell lines 697 and 107.2, respectively. B, Stage distribution of huλ5 and mλ5 mRNA in huλ5 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 huλ5 and mλ5 expression, respectively. Data shown in this panel were from films exposed for 17.5 h for huλ5 (including 697), 2 h for mλ5 (including 107.2), and 0.5 h for GAPDH. Calculations for relative huλ5 and mλ5 expression and for pro B/pre B vs B expression were done with consistent 0.5 h exposures and analyzed on a phosphoimager. Huλ5 and mλ5 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 huλ5 nor mλ5 were expressed in these populations (data not shown).

Although transgenic huλ5 transcripts are present in the thymus, the huλ5 polypeptide was not detected in thymocytes by flow cytometric analysis (data not shown). The ratio of CD4 and CD8 subsets appeared normal (data not shown), indicating that huλ5 mRNA expression does not interfere with normal mouse thymus development. Taken together, the presence of the 28-kb huλ5 transgene confers huλ5 expression at the RNA level, suggesting this genomic fragment contains the necessary cis-regulatory elements for transcription in the bone marrow, thymus, and testis.

Huλ5 expression is limited to the pro-B/pre-B lymphocyte fraction within the B lineage

To determine whether the huλ5 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 huλ5 transgenic (mλ5^+/+) mouse. RT-PCR was performed with poly(A)⁺-mRNA from the above B lymphoid fractions of a huλ5 transgenic mouse, and PCR products were analyzed on Southern blots using the 0.18-kb BglI huλ5-specific probe. The transgenic huλ5 gene is predominately expressed in the pro-B/pre-B populations (B220⁺, sIgM⁻) in the bone marrow (Fig. 2⇑B). In addition, expression of the mλ5 gene was restricted to the same B lymphoid cell population (Fig. 2⇑B). After normalizing the huλ5 and mλ5 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 huλ5 (at 1:16 and 1:4 dilutions, respectively) and about 10.2 times more mλ5 (at the 1:4 dilution) mRNA transcripts as does the B cell population (B220⁺, sIgM⁺). This indicates that expression of huλ5, as has been previously shown for mλ5, (3, 4, 5, 27, 28) is largely restricted to the pro-B/pre-B cell populations. These results are consistent with the huλ5 transgene being expressed in a stage-specific fashion within the B cell lineage. To compare huλ5 and mλ5 expression within the pro-B/pre-B population (after multiplying the huλ5 signal by three to take into account its smaller fragment size), mλ5 is 2.8–3.4 times higher than huλ5 expression (1:16 and 1:4 dilutions, respectively).

The huλ5 protein is expressed in a stage-specific fashion in huλ5 transgenic mice

To determine whether the huλ5 transgene is expressed at the protein level, we membrane-stained ex vivo bone marrow cells from transgenic huλ5^+/− and nontransgenic mλ5^+/+ 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 huλ5 with the monoclonal mouse IgG1 anti-human λ5 Ab, HSL11 (55), followed by the FITC-conjugated rat anti-mouse γ1 Ab. The data presented in Fig. 3⇓ reveal that CD43⁺ B220^low pro-B/early pre-B cells (Fig. 3⇓B), as well as the CD43⁻ B220^low pre-B/immature B cells (Fig. 3⇓C) from transgenic huλ5 mice stain positively, whereas pro-B/early pre-B cells from nontransgenic mλ5 ^+/+ wild-type littermates did not react with the anti-huλ5 Ab (Fig. 3⇓F). In addition, as expected from the huλ5 transgenic RNA expression studies (Fig. 2⇑), CD43⁻ B220^high mature B cells from huλ5 transgenic bone marrow also fail to react with the monoclonal anti-huλ5 Ab (Fig. 3⇓D). Attempts to detect huλ5 on the surface of ex vivo bone marrow cells failed as has been reported by others for mλ5 (58). We conclude from these data that the huλ5 transgene is expressed at the RNA and protein levels in a stage-specific fashion.

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FIGURE 3.

Flow cytometric analysis of huλ5 protein expression in ex vivo huλ5 transgenic mice bone marrow cells. Bone marrow cells from a huλ5^+/− transgene on a wild-type mλ5^+/+ background (A–D) or wild-type mλ5^+/+ (E and F) mice were stained for membrane CD43 and B220. Cells were fixed, permeablilized, and cytoplasmically stained for huλ5 with HSL11 and rat anti-mouse γ1-FITC. A, Membrane CD43 and B220 staining on bone marrow cells prepared from a huλ5^+/−mλ5^+/+ mouse. The percentage of pro-B/early pre-B, (R2), pre-B/immature-B (R3), and mature B (R4) are shown. B, Percentage of huλ5 protein expressed cytoplasmically in the gated pro-B/early pre-B (R2) bone marrow cell subpopulations from A. C, Percentage of huλ5 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-huλ5 Ab. E, Membrane CD43 and B220 staining on bone marrow cells prepared from a wild-type (mλ5^+/+) mouse. Gated B lineage populations are depicted in the boxes as in A. F, Huλ5 cytoplasmic staining of the wild-type mouse pro-B/early pre-B cell population (R2 of E).

The huλ5 transgene rescues the mλ5 null phenotype

One line of huλ5 transgenic mice was crossed with mλ5 nullizygous mλ5^−/− mice to investigate whether the huλ5 transgene could rescue the null phenotype. Offspring were analyzed for the presence of the huλ5 transgene and the genotype at the mλ5 locus as described in Materials and Methods. Flow cytometric analyses performed on bone marrow cells from transgenic heterozygous huλ5 (huλ5^+/−mλ5^+/+) 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. 4⇓A). In contrast, the nontransgenic mλ5^−/− 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. 4⇓B). In huλ5 transgenic mice with homozygous deletions at the mλ5 gene, mλ5^−/−, bone marrow cells have increased numbers of CD43⁻/B220⁺ pre-B/immature B cells (9.9%) and CD43⁻/B220⁺ B cells (5.8%) (Fig. 4⇓C) similar to that observed in huλ5^+/−mλ5^+/+ 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 huλ5 transgene is able to reconstitute the block at the CD43⁺/B220⁺ stage in the mλ5^−/− mice.

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FIGURE 4.

Flow cytometric analysis of B lineage markers on bone marrow cells from 8-wk-old huλ5^+/−mλ5^+/+ (A), mλ5^−/− (B), and huλ5^+/−mλ5^−/− 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 huλ5 transgenic mice produce a huλ5 protein that is expressed on the cell surface and binds mVpreB and μHC

Because the huλ5 transgene rescued the block at the CD43⁺/B220⁺ stage in mλ5 nullizygous mice, we expected to detect mouse μ and huλ5 chains on the surface of A-MuLV-transformed pre-B cell lines prepared from huλ5^+/−mλ5^−/− mice. A priori, we expected to detect huλ5 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 huλ5^+/−mλ5^−/− 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 mλ5 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 huλ5, 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 huλ5 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. 5⇓A) at levels comparable to that of the A-MuLV-transformed pre-B cell lines 107.2 (Fig. 5⇓B) or TK.μ (data not shown) (20). H9 does express membrane huλ5 (Fig. 5⇓C), whereas the control (wild-type) 107.2 cell line does not (<1%) (Fig. 5⇓D). As expected, Abs against mλ5 react only with 107.2 cells (Fig. 5⇓F) but not with Hula cells (Fig. 5⇓E). Because others have shown that mμ chains that fail to pair with λ5 chains are not transported to the cell surface (20, 22) and full-length mμ requires mλ5 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 huλ5 protein is able to carry the mμ and most likely mVpreB to the cell surface.

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FIGURE 5.

Membrane fluorescent staining of huλ5^+/−mλ5^−/− Hula lines. A-MuLV-transformed cell lines were prepared from bone marrow cells from the huλ5^+/−mλ5^−/− mice. Hula line H9 (38A1 (5 ) H9) was analyzed for synthesis of membrane mμ (A), huλ5 (C), mλ5 (E), and murine κ (G) and compared with the mouse Abelson line, 107.2, known to be positive for mμ, mλ5, and κ. The Hula H9 cells are positive for membrane staining of mμ (A) and huλ5 (C), but not for κ (G). The 107.2 cells are positive for mμ (B), mλ5 (F), and mκ (H), but negative for staining with the anti-huλ5 (HSL11) Ab (D).

To address whether the huλ5 protein associates with mμ and mVpreB in the Hula huλ5^+/−mλ5^−/− pre-B cell lines, we performed immunoprecipitation assays. Immunoprecipitation of H9 and H11 A-MuLV-transformed pre-B cell lines using anti-mμ shows mVpreB and huλ5 at 16 and 21 kDa, respectively (Fig. 6⇓A, lanes 2 and 3). The μ-positive human pre-B acute lymphocytic leukemic cell line, Nalm-6, shows coimmunoprecipitation of huλ5 and huVpreB at 21 and 18 kDa, respectively (Fig. 6⇓A, lane 1). The mouse A-MuLV pre-B cell line, TKμ, coimmunoprecipitates mλ5 and mVpreB at 22 and 16 kDa, respectively (Fig. 6⇓A, lane 4). Results from multiple experiments show that there is a different molecular mass between huλ5 and mλ5 (18 vs 16 kDa, respectively). Secondary mouse anti-rat IgG alone did not show specific bands precipitated (Fig. 6⇓A, lanes 5–7). Hula huλ5^+/−mλ5^−/− subclones H10 and H9 were also immunoprecipitated with anti-huλ5 Abs and showed coprecipitation of mVpreB and mμ (Fig. 6⇓B, lanes 1–3). Immunoprecipitation of the Nalm-6 cell line shows that the anti-huλ5 Ab coprecipitates huμ and huVpreB (Fig. 6⇓B, lanes 4 and 8). In contrast, anti-huλ5 Abs do not coprecipitate mμ or mVpreB from the Tκμ A-MuLV pre-B cell line (Fig. 6⇓B, lane 5). These results show that the huλ5 protein can associate with mVpreB and mμ into a chimeric pre-BCR in pre-B cell lines prepared from the bone marrow of huλ5^+/−mλ5^−/− mice, although this association appears to be weaker (or less stable) than that of the native huλ5/huVpreB/huμ seen in Nalm-6 (see Discussion). The differences in size of the mμ band in different lines (see for example H9, lane 2, and H11, lane 3, Fig. 6⇓A) may be due to differential glycosylation, although this has not been formally tested. We do not believe that huλ5 is secreted in the Abelson lines, as supernatants were negative for anti-huλ5 precipitation.

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FIGURE 6.

Analysis of immunoprecipitated mouse and human pre-BCR in A-MuLV-transformed cells lines prepared from huλ5^+/−mλ5^−/− mice (Hula cell lines). Mouse and human pre-BCR (μ, λ5, VpreB) were analyzed in ³⁵S-labeled cellular extracts from Hula lines, the μ-positive mouse A-MuLV pre-B cell line, Tκμ, and the human pre-B acute lymphoblastic leukemia line, Nalm-6, in anti-μ and anti-huλ5 precipitates. A, Anti-mμ precipitation of huλ5 and mVpreB in Nalm-6 (lane 1), Hula lines H9 and H11 (lanes 2 and 3), and Tκμ (lane 4). Secondary (2°) Ab (mouse anti-rat IgG) alone showed no specific bands precipitated (lanes 5–7). B, Anti-huλ5 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, Tκμ (lane 5), was used as a negative control for the anti-huλ5 Ab. Data from two different huλ5^+/−mλ5^−/− mice (H for H9, H10, H11; 45 for 45.1) are shown.