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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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5′λ5 is active in BM but not in spleen, thymus, heart, muscle,  or liver. 5′λ5 hu-CD25: 5′λ5 inserted upstream of the cDNA encoding  human IL-2–R (hu-CD25; reference 21) followed by exon 2, intron 2,  exon 3 (solid boxes), and polyA site (hatched box) from human β-globin  (11). The 5′λ5 hu-CD25 was used to establish transgenic mice. (A) BM,  spleen, and thymus cells were analyzed by FACS® using antibodies detecting mouse CD19 or CD4 and hu-CD25. Data from nontransgenic  (tg−) and transgenic (tg+) littermates from founder 82 are shown. (B) Total  RNA from heart, muscle, liver, spleen, and BM was isolated and analyzed  for expression of hu-CD25 and HPRT by RT-PCR analysis. Arrows denote the 185 and 220-bp fragments of hu-CD25 or HPRT, respectively.
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Figure 1: 5′λ5 is active in BM but not in spleen, thymus, heart, muscle, or liver. 5′λ5 hu-CD25: 5′λ5 inserted upstream of the cDNA encoding human IL-2–R (hu-CD25; reference 21) followed by exon 2, intron 2, exon 3 (solid boxes), and polyA site (hatched box) from human β-globin (11). The 5′λ5 hu-CD25 was used to establish transgenic mice. (A) BM, spleen, and thymus cells were analyzed by FACS® using antibodies detecting mouse CD19 or CD4 and hu-CD25. Data from nontransgenic (tg−) and transgenic (tg+) littermates from founder 82 are shown. (B) Total RNA from heart, muscle, liver, spleen, and BM was isolated and analyzed for expression of hu-CD25 and HPRT by RT-PCR analysis. Arrows denote the 185 and 220-bp fragments of hu-CD25 or HPRT, respectively.

Mentions: The analysis of the cellularity as well as the staining of the different B cell differentiation stages in BM and spleen (as described in detail previously in reference 15) revealed that in none of the transgenic lines was lymphocyte development disturbed by any means (Table 2) in agreement with other studies using hu-CD25 as transgene (22). All three founder strains expressing the transgene showed similar staining patterns in BM (as shown in Fig. 1 A for the founder 82). Several levels (high, intermediate, and low) of hu-CD25 expression were detected in B lineage cells in the BM, while spleen and thymus cells were practically negative (Fig. 1 A). The ∼0.5% of spleen cells which expressed very low levels of hu-CD25 were found to be IgM+IgD− immature B cells (data not shown). Most hu-CD25 positive BM cells expressed the B cell markers CD19 and CD45R (B220; Fig. 1 A, and data not shown). RT-PCR analysis of BM, spleen, liver, muscle, and heart readily detected huCD25 RNA in the BM and low levels in the spleen but no signal was found in the nonlymphoid samples, while the HPRT control primers detected HPRT RNA in all organs (Fig. 1 B). Thus, the 5′λ5 control region contains lineage– and differentiation stage–specific promoter/enhancer activity.


A transgenic marker for mouse B lymphoid precursors.

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

5′λ5 is active in BM but not in spleen, thymus, heart, muscle,  or liver. 5′λ5 hu-CD25: 5′λ5 inserted upstream of the cDNA encoding  human IL-2–R (hu-CD25; reference 21) followed by exon 2, intron 2,  exon 3 (solid boxes), and polyA site (hatched box) from human β-globin  (11). The 5′λ5 hu-CD25 was used to establish transgenic mice. (A) BM,  spleen, and thymus cells were analyzed by FACS® using antibodies detecting mouse CD19 or CD4 and hu-CD25. Data from nontransgenic  (tg−) and transgenic (tg+) littermates from founder 82 are shown. (B) Total  RNA from heart, muscle, liver, spleen, and BM was isolated and analyzed  for expression of hu-CD25 and HPRT by RT-PCR analysis. Arrows denote the 185 and 220-bp fragments of hu-CD25 or HPRT, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: 5′λ5 is active in BM but not in spleen, thymus, heart, muscle, or liver. 5′λ5 hu-CD25: 5′λ5 inserted upstream of the cDNA encoding human IL-2–R (hu-CD25; reference 21) followed by exon 2, intron 2, exon 3 (solid boxes), and polyA site (hatched box) from human β-globin (11). The 5′λ5 hu-CD25 was used to establish transgenic mice. (A) BM, spleen, and thymus cells were analyzed by FACS® using antibodies detecting mouse CD19 or CD4 and hu-CD25. Data from nontransgenic (tg−) and transgenic (tg+) littermates from founder 82 are shown. (B) Total RNA from heart, muscle, liver, spleen, and BM was isolated and analyzed for expression of hu-CD25 and HPRT by RT-PCR analysis. Arrows denote the 185 and 220-bp fragments of hu-CD25 or HPRT, respectively.
Mentions: The analysis of the cellularity as well as the staining of the different B cell differentiation stages in BM and spleen (as described in detail previously in reference 15) revealed that in none of the transgenic lines was lymphocyte development disturbed by any means (Table 2) in agreement with other studies using hu-CD25 as transgene (22). All three founder strains expressing the transgene showed similar staining patterns in BM (as shown in Fig. 1 A for the founder 82). Several levels (high, intermediate, and low) of hu-CD25 expression were detected in B lineage cells in the BM, while spleen and thymus cells were practically negative (Fig. 1 A). The ∼0.5% of spleen cells which expressed very low levels of hu-CD25 were found to be IgM+IgD− immature B cells (data not shown). Most hu-CD25 positive BM cells expressed the B cell markers CD19 and CD45R (B220; Fig. 1 A, and data not shown). RT-PCR analysis of BM, spleen, liver, muscle, and heart readily detected huCD25 RNA in the BM and low levels in the spleen but no signal was found in the nonlymphoid samples, while the HPRT control primers detected HPRT RNA in all organs (Fig. 1 B). Thus, the 5′λ5 control region contains lineage– and differentiation stage–specific promoter/enhancer activity.

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