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Developmental separation of V(D)J recombinase expression and initiation of IgH recombination in B lineage progenitors in vivo.

Borghesi L, Gerstein RM - J. Exp. Med. (2004)

Bottom Line: To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors.We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable.Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts Medical School, Molecular Genetics and Microbiology, 55 Lake Ave. North, Worcester 01655, USA.

ABSTRACT
In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

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V(D)J recombinase activity in early B lineage precursors. (A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. (B) Bone marrow from SB110 H2-SVEX transgenic mice was stained with antibodies to Ly6C, DX5, B220, CD43, CD19, CD24, and IgM in order to examine VEX expression throughout B cell development. The percentage of cells in each region is given. The data are representative of three independent experiments.
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fig1: V(D)J recombinase activity in early B lineage precursors. (A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. (B) Bone marrow from SB110 H2-SVEX transgenic mice was stained with antibodies to Ly6C, DX5, B220, CD43, CD19, CD24, and IgM in order to examine VEX expression throughout B cell development. The percentage of cells in each region is given. The data are representative of three independent experiments.

Mentions: The H2-SVEX transgene was constructed by placing the RSS-VEX-RSS fragment (Fig. 1 A) into the H2K (HIL) transgenic vector using a unique NotI restriction site located between the H2 promoter and the H2 exon fragment as described previously (21). The H2K cassette vector expresses genes under the control of the H2K promoter/enhancer and Moloney MuLV enhancer/poly(A), typically at high levels in HSC and all hematolymphoid cells (21). Inbred C57BL/6 transgenic mice were generated using standard procedures (21).


Developmental separation of V(D)J recombinase expression and initiation of IgH recombination in B lineage progenitors in vivo.

Borghesi L, Gerstein RM - J. Exp. Med. (2004)

V(D)J recombinase activity in early B lineage precursors. (A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. (B) Bone marrow from SB110 H2-SVEX transgenic mice was stained with antibodies to Ly6C, DX5, B220, CD43, CD19, CD24, and IgM in order to examine VEX expression throughout B cell development. The percentage of cells in each region is given. The data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211822&req=5

fig1: V(D)J recombinase activity in early B lineage precursors. (A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. (B) Bone marrow from SB110 H2-SVEX transgenic mice was stained with antibodies to Ly6C, DX5, B220, CD43, CD19, CD24, and IgM in order to examine VEX expression throughout B cell development. The percentage of cells in each region is given. The data are representative of three independent experiments.
Mentions: The H2-SVEX transgene was constructed by placing the RSS-VEX-RSS fragment (Fig. 1 A) into the H2K (HIL) transgenic vector using a unique NotI restriction site located between the H2 promoter and the H2 exon fragment as described previously (21). The H2K cassette vector expresses genes under the control of the H2K promoter/enhancer and Moloney MuLV enhancer/poly(A), typically at high levels in HSC and all hematolymphoid cells (21). Inbred C57BL/6 transgenic mice were generated using standard procedures (21).

Bottom Line: To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors.We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable.Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts Medical School, Molecular Genetics and Microbiology, 55 Lake Ave. North, Worcester 01655, USA.

ABSTRACT
In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

Show MeSH