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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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FACS® sorting procedure and reanalyzed data of  transgenic BM cells analyzed for  expression of B220, CD19, CD4,  NK1.1, and hu-CD25. BM cells  depleted of IgM+ cells (see Materials and Methods) were stained  with anti–B220-APC, anti–CD19PE, anti–CD4-PE, anti–NK1.1PE, and anti–hu-CD25–FITC.  B220+CD19−CD4−NK1.1− cells  (gate R1) were gated and analyzed for hu-CD25 expression.  Hu-CD25 positive (gate R3) and  negative cells (gate R2) among  the B220+CD19− CD4−NK1.1−  population were sorted separately and the obtained fractions  were reanalyzed after sorting.
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Figure 4: FACS® sorting procedure and reanalyzed data of transgenic BM cells analyzed for expression of B220, CD19, CD4, NK1.1, and hu-CD25. BM cells depleted of IgM+ cells (see Materials and Methods) were stained with anti–B220-APC, anti–CD19PE, anti–CD4-PE, anti–NK1.1PE, and anti–hu-CD25–FITC. B220+CD19−CD4−NK1.1− cells (gate R1) were gated and analyzed for hu-CD25 expression. Hu-CD25 positive (gate R3) and negative cells (gate R2) among the B220+CD19− CD4−NK1.1− population were sorted separately and the obtained fractions were reanalyzed after sorting.

Mentions: DJH rearrangements of the H chain locus were amplified and detected using PCR as outlined in Fig. 4. Two forward primers binding immediately upstream of DFL/DSP elements or the DQ52 element, respectively, were used in a mixture (DFS and DQ52 as described in reference 20) together with one reverse primer binding downstream of JH4 (JH4A in reference 16). In germline configuration, the DQ52 and JH4A primers will amplify the 2.15-kb germline fragment. DJH1, DJH2, DJH3, and DJH4 rearrangements involving either DFL, DSP, or DQ52 elements will be detected by the emergence of bands of 1.46, 1.15, 0.73, and 0.20 kb, respectively.


A transgenic marker for mouse B lymphoid precursors.

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

FACS® sorting procedure and reanalyzed data of  transgenic BM cells analyzed for  expression of B220, CD19, CD4,  NK1.1, and hu-CD25. BM cells  depleted of IgM+ cells (see Materials and Methods) were stained  with anti–B220-APC, anti–CD19PE, anti–CD4-PE, anti–NK1.1PE, and anti–hu-CD25–FITC.  B220+CD19−CD4−NK1.1− cells  (gate R1) were gated and analyzed for hu-CD25 expression.  Hu-CD25 positive (gate R3) and  negative cells (gate R2) among  the B220+CD19− CD4−NK1.1−  population were sorted separately and the obtained fractions  were reanalyzed after sorting.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: FACS® sorting procedure and reanalyzed data of transgenic BM cells analyzed for expression of B220, CD19, CD4, NK1.1, and hu-CD25. BM cells depleted of IgM+ cells (see Materials and Methods) were stained with anti–B220-APC, anti–CD19PE, anti–CD4-PE, anti–NK1.1PE, and anti–hu-CD25–FITC. B220+CD19−CD4−NK1.1− cells (gate R1) were gated and analyzed for hu-CD25 expression. Hu-CD25 positive (gate R3) and negative cells (gate R2) among the B220+CD19− CD4−NK1.1− population were sorted separately and the obtained fractions were reanalyzed after sorting.
Mentions: DJH rearrangements of the H chain locus were amplified and detected using PCR as outlined in Fig. 4. Two forward primers binding immediately upstream of DFL/DSP elements or the DQ52 element, respectively, were used in a mixture (DFS and DQ52 as described in reference 20) together with one reverse primer binding downstream of JH4 (JH4A in reference 16). In germline configuration, the DQ52 and JH4A primers will amplify the 2.15-kb germline fragment. DJH1, DJH2, DJH3, and DJH4 rearrangements involving either DFL, DSP, or DQ52 elements will be detected by the emergence of bands of 1.46, 1.15, 0.73, and 0.20 kb, respectively.

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