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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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Downregulation of transgene expression upon differentiation of in vitro cultivated pre–B cell lines. Pre–B cell lines were cultivated in the  presence of IL-7 on ST-2 stromal cells as described (15), and differentiation was induced by withdrawal of IL-7. (A) After 0, 1, 2, and 3 d in the absence  of IL-7, the cells were stained with antibodies specific for CD19, λ5 (as detected by the mAb LM34; reference 23), hu-CD25, and IgM, and analyzed by  FACS®. Fluorescence intensities are displayed (solid histograms) and the fluorescence intensity of an irrelevant antibody is overlaid (open histograms). (B) After 0, 1, 2, 3, and 5 d in the absence of IL-7, the cells were harvested and RNA was prepared and subjected to Northern blot analysis using λ5, hu-CD25,  and β-actin specific probes.
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Figure 3: Downregulation of transgene expression upon differentiation of in vitro cultivated pre–B cell lines. Pre–B cell lines were cultivated in the presence of IL-7 on ST-2 stromal cells as described (15), and differentiation was induced by withdrawal of IL-7. (A) After 0, 1, 2, and 3 d in the absence of IL-7, the cells were stained with antibodies specific for CD19, λ5 (as detected by the mAb LM34; reference 23), hu-CD25, and IgM, and analyzed by FACS®. Fluorescence intensities are displayed (solid histograms) and the fluorescence intensity of an irrelevant antibody is overlaid (open histograms). (B) After 0, 1, 2, 3, and 5 d in the absence of IL-7, the cells were harvested and RNA was prepared and subjected to Northern blot analysis using λ5, hu-CD25, and β-actin specific probes.

Mentions: The developmental control of transgenic hu-CD25 expression in B lymphocyte development was further analyzed in differentiating pre–B cells in vitro. c-kit+ pre–BI cells can be expanded in vitro on stromal cells in the presence of IL-7 (15). Therefore, c-kit+ pre–BI cells from fetal liver of transgenic mice were cultured on ST-2 stromal cells in the presence of IL-7 and transduced with a retrovirus encoding the bcl-2 cDNA to inhibit apoptosis after removal of IL-7. As shown in Fig. 3 A, the cells in culture expressed CD19, λ5 protein (as detected by the LM34 antibody; reference 23) and high levels of hu-CD25, but no detectable IgM on the cell surface. Withdrawal of IL-7 and analysis 1, 2, and 3 d thereafter demonstrated that the level of CD19 expression remained constant while the surface expression of λ5 and hu-CD25 decreased with time. In addition, ∼10% of the cells started to express IgM on the cell surface at day 3 of differentiation. Furthermore, Northern blot analysis of these differentiating cells showed that the RNA steady state levels of hu-CD25 and λ5 decreased with time of differentiation (Fig. 3 B). Disappearance of hu-CD25 paralleled the disappearance of λ5 RNA (Fig. 3 B). Again, these results indicate that the hu-CD25 transgene, like endogenous λ5, is expressed in pre–BI cells. Expression of the endogenous λ5 and hu-CD25 transgene is downregulated as these pre–BI cells differentiate in vitro to sIgM+ immature B cells.


A transgenic marker for mouse B lymphoid precursors.

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

Downregulation of transgene expression upon differentiation of in vitro cultivated pre–B cell lines. Pre–B cell lines were cultivated in the  presence of IL-7 on ST-2 stromal cells as described (15), and differentiation was induced by withdrawal of IL-7. (A) After 0, 1, 2, and 3 d in the absence  of IL-7, the cells were stained with antibodies specific for CD19, λ5 (as detected by the mAb LM34; reference 23), hu-CD25, and IgM, and analyzed by  FACS®. Fluorescence intensities are displayed (solid histograms) and the fluorescence intensity of an irrelevant antibody is overlaid (open histograms). (B) After 0, 1, 2, 3, and 5 d in the absence of IL-7, the cells were harvested and RNA was prepared and subjected to Northern blot analysis using λ5, hu-CD25,  and β-actin specific probes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196144&req=5

Figure 3: Downregulation of transgene expression upon differentiation of in vitro cultivated pre–B cell lines. Pre–B cell lines were cultivated in the presence of IL-7 on ST-2 stromal cells as described (15), and differentiation was induced by withdrawal of IL-7. (A) After 0, 1, 2, and 3 d in the absence of IL-7, the cells were stained with antibodies specific for CD19, λ5 (as detected by the mAb LM34; reference 23), hu-CD25, and IgM, and analyzed by FACS®. Fluorescence intensities are displayed (solid histograms) and the fluorescence intensity of an irrelevant antibody is overlaid (open histograms). (B) After 0, 1, 2, 3, and 5 d in the absence of IL-7, the cells were harvested and RNA was prepared and subjected to Northern blot analysis using λ5, hu-CD25, and β-actin specific probes.
Mentions: The developmental control of transgenic hu-CD25 expression in B lymphocyte development was further analyzed in differentiating pre–B cells in vitro. c-kit+ pre–BI cells can be expanded in vitro on stromal cells in the presence of IL-7 (15). Therefore, c-kit+ pre–BI cells from fetal liver of transgenic mice were cultured on ST-2 stromal cells in the presence of IL-7 and transduced with a retrovirus encoding the bcl-2 cDNA to inhibit apoptosis after removal of IL-7. As shown in Fig. 3 A, the cells in culture expressed CD19, λ5 protein (as detected by the LM34 antibody; reference 23) and high levels of hu-CD25, but no detectable IgM on the cell surface. Withdrawal of IL-7 and analysis 1, 2, and 3 d thereafter demonstrated that the level of CD19 expression remained constant while the surface expression of λ5 and hu-CD25 decreased with time. In addition, ∼10% of the cells started to express IgM on the cell surface at day 3 of differentiation. Furthermore, Northern blot analysis of these differentiating cells showed that the RNA steady state levels of hu-CD25 and λ5 decreased with time of differentiation (Fig. 3 B). Disappearance of hu-CD25 paralleled the disappearance of λ5 RNA (Fig. 3 B). Again, these results indicate that the hu-CD25 transgene, like endogenous λ5, is expressed in pre–BI cells. Expression of the endogenous λ5 and hu-CD25 transgene is downregulated as these pre–BI cells differentiate in vitro to sIgM+ immature B cells.

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.

Show MeSH
Related in: MedlinePlus