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A transgenic marker for mouse B lymphoid precursors.

Mårtensson IL, Melchers F, Winkler TH - J. Exp. Med. (1997)

Bottom Line: All three strains express human CD25 in parallel to endogenous lambda5 on pre-B cells, but not on mature B lymphocytes or other blood cell lineages.Both types of precursors are clonable on stromal cells in the presence of interleukin-7.The CD19- precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre-BI cells are predominantly DJH rearranged.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, Immunology Group, Lund, Sweden.

ABSTRACT
Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5' of the precursor (pre)-B cell-specific lambda5 gene. All three strains express human CD25 in parallel to endogenous lambda5 on pre-B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220+CD19-lambda5+ precursor of the B220+CD19+lambda5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19- precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre-BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5' region of the lambda5 gene into the mouse genome confers tissue and differentiation stage-specific expression of a transgene.

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Characterization of the B220+CD19− negative cell population in BM of mice expressing the hu-CD25 transgene under the control of the  5′λ5. (A) Expression of hu-CD25, endogenous λ5, and HPRT in sorted cell populations. BM cells were sorted by FACS® after triple labeling for B220,  CD19, and hu-CD25, or double labeling for CD19 and c-kit, respectively. Fivefold dilutions of cDNA from sorted cells were subjected to PCR amplification of hu-CD25, λ5, and HPRT genes and PCR products detected by Southern blotting with specific probes. (B) PCR-mediated analysis of the rearrangement status of the IgH locus in sorted cell populations. DNA of sorted cells was subjected to PCR amplification as outlined in the top of the figure. After electrophoresis and Southern blotting, the PCR products were detected by hybridization with a JH4-specific oligonucleotide. Sorted cell  populations are in lane 1, B220− c-kithigh; lane 2, CD19+ c-kit+, and lane 3, B220+CD19− hu-CD25+. (C) Limiting dilution analysis of FACS® sorted  B220+ cells from BM on ST-2 stromal cells in the presence of IL-7. Growth of pre–B cell colonies consisting of >50 cells was scored on day 7 of culture.  24 replicate cultures for each cell number were initiated. According to Poisson distribution, one precursor is present in the cell suspension when 37% of  the cultures do not show colonies.
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Figure 5: Characterization of the B220+CD19− negative cell population in BM of mice expressing the hu-CD25 transgene under the control of the 5′λ5. (A) Expression of hu-CD25, endogenous λ5, and HPRT in sorted cell populations. BM cells were sorted by FACS® after triple labeling for B220, CD19, and hu-CD25, or double labeling for CD19 and c-kit, respectively. Fivefold dilutions of cDNA from sorted cells were subjected to PCR amplification of hu-CD25, λ5, and HPRT genes and PCR products detected by Southern blotting with specific probes. (B) PCR-mediated analysis of the rearrangement status of the IgH locus in sorted cell populations. DNA of sorted cells was subjected to PCR amplification as outlined in the top of the figure. After electrophoresis and Southern blotting, the PCR products were detected by hybridization with a JH4-specific oligonucleotide. Sorted cell populations are in lane 1, B220− c-kithigh; lane 2, CD19+ c-kit+, and lane 3, B220+CD19− hu-CD25+. (C) Limiting dilution analysis of FACS® sorted B220+ cells from BM on ST-2 stromal cells in the presence of IL-7. Growth of pre–B cell colonies consisting of >50 cells was scored on day 7 of culture. 24 replicate cultures for each cell number were initiated. According to Poisson distribution, one precursor is present in the cell suspension when 37% of the cultures do not show colonies.

Mentions: The marker-negative subpopulation was sorted into huCD25+ and hu-CD25− cells and analyzed for other markers, in particular for the expression of endogenous λ5. RTPCR analysis shown in Fig. 5 A revealed that these cells, like CD19+ pre–BI cells coexpressed hu-CD25 and endogenous λ5, whereas hu-CD25− sorted cells from the same B220+CD19− population contained no detectable λ5 or hu-CD25 message. The B220+CD19+ hu-CD25+ cells also expressed VpreB, sterile μ H chain, RAG-1, RAG-2, and B29 transcripts (data not shown). PCR analysis of the H chain gene loci of the B220+CD19− hu-CD25+ cells revealed that a sizeable fraction of all loci were still in germline configuration while the rest of these loci were DJH rearranged (Fig. 5 B, lane 3). VHDJH rearranged loci could not be detected (data not shown). In CD19+ c-kit+ pre–BI cells (lane 2) the H chain gene loci were found to be mostly DJH rearranged in agreement with previous analyses (9).


A transgenic marker for mouse B lymphoid precursors.

Mårtensson IL, Melchers F, Winkler TH - J. Exp. Med. (1997)

Characterization of the B220+CD19− negative cell population in BM of mice expressing the hu-CD25 transgene under the control of the  5′λ5. (A) Expression of hu-CD25, endogenous λ5, and HPRT in sorted cell populations. BM cells were sorted by FACS® after triple labeling for B220,  CD19, and hu-CD25, or double labeling for CD19 and c-kit, respectively. Fivefold dilutions of cDNA from sorted cells were subjected to PCR amplification of hu-CD25, λ5, and HPRT genes and PCR products detected by Southern blotting with specific probes. (B) PCR-mediated analysis of the rearrangement status of the IgH locus in sorted cell populations. DNA of sorted cells was subjected to PCR amplification as outlined in the top of the figure. After electrophoresis and Southern blotting, the PCR products were detected by hybridization with a JH4-specific oligonucleotide. Sorted cell  populations are in lane 1, B220− c-kithigh; lane 2, CD19+ c-kit+, and lane 3, B220+CD19− hu-CD25+. (C) Limiting dilution analysis of FACS® sorted  B220+ cells from BM on ST-2 stromal cells in the presence of IL-7. Growth of pre–B cell colonies consisting of >50 cells was scored on day 7 of culture.  24 replicate cultures for each cell number were initiated. According to Poisson distribution, one precursor is present in the cell suspension when 37% of  the cultures do not show colonies.
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Figure 5: Characterization of the B220+CD19− negative cell population in BM of mice expressing the hu-CD25 transgene under the control of the 5′λ5. (A) Expression of hu-CD25, endogenous λ5, and HPRT in sorted cell populations. BM cells were sorted by FACS® after triple labeling for B220, CD19, and hu-CD25, or double labeling for CD19 and c-kit, respectively. Fivefold dilutions of cDNA from sorted cells were subjected to PCR amplification of hu-CD25, λ5, and HPRT genes and PCR products detected by Southern blotting with specific probes. (B) PCR-mediated analysis of the rearrangement status of the IgH locus in sorted cell populations. DNA of sorted cells was subjected to PCR amplification as outlined in the top of the figure. After electrophoresis and Southern blotting, the PCR products were detected by hybridization with a JH4-specific oligonucleotide. Sorted cell populations are in lane 1, B220− c-kithigh; lane 2, CD19+ c-kit+, and lane 3, B220+CD19− hu-CD25+. (C) Limiting dilution analysis of FACS® sorted B220+ cells from BM on ST-2 stromal cells in the presence of IL-7. Growth of pre–B cell colonies consisting of >50 cells was scored on day 7 of culture. 24 replicate cultures for each cell number were initiated. According to Poisson distribution, one precursor is present in the cell suspension when 37% of the cultures do not show colonies.
Mentions: The marker-negative subpopulation was sorted into huCD25+ and hu-CD25− cells and analyzed for other markers, in particular for the expression of endogenous λ5. RTPCR analysis shown in Fig. 5 A revealed that these cells, like CD19+ pre–BI cells coexpressed hu-CD25 and endogenous λ5, whereas hu-CD25− sorted cells from the same B220+CD19− population contained no detectable λ5 or hu-CD25 message. The B220+CD19+ hu-CD25+ cells also expressed VpreB, sterile μ H chain, RAG-1, RAG-2, and B29 transcripts (data not shown). PCR analysis of the H chain gene loci of the B220+CD19− hu-CD25+ cells revealed that a sizeable fraction of all loci were still in germline configuration while the rest of these loci were DJH rearranged (Fig. 5 B, lane 3). VHDJH rearranged loci could not be detected (data not shown). In CD19+ c-kit+ pre–BI cells (lane 2) the H chain gene loci were found to be mostly DJH rearranged in agreement with previous analyses (9).

Bottom Line: All three strains express human CD25 in parallel to endogenous lambda5 on pre-B cells, but not on mature B lymphocytes or other blood cell lineages.Both types of precursors are clonable on stromal cells in the presence of interleukin-7.The CD19- precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre-BI cells are predominantly DJH rearranged.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, Immunology Group, Lund, Sweden.

ABSTRACT
Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5' of the precursor (pre)-B cell-specific lambda5 gene. All three strains express human CD25 in parallel to endogenous lambda5 on pre-B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220+CD19-lambda5+ precursor of the B220+CD19+lambda5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19- precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre-BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5' region of the lambda5 gene into the mouse genome confers tissue and differentiation stage-specific expression of a transgene.

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