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hCD2-iCre and Vav-iCre mediated gene recombination patterns in murine hematopoietic cells.

Siegemund S, Shepherd J, Xiao C, Sauer K - PLoS ONE (2015)

Bottom Line: R26-stop-EYFP ubiquitously encodes EYFP preceded by a floxed stop cassette.By removing it, Cre activity induces measurable EYFP expression.Our results confirm the known activity patterns for both Cre transgenes and unveil additional hCD2-iCre mediated reporter gene recombination in common lymphoid progenitors, in natural killer cells and their progenitors, and in plasmacytoid and conventional dendritic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
Cre-recombinase mediated conditional deletion of Lox-P site flanked ("floxed") genes is widely used for functional gene annotation in mice. Many different Cre-transgenic mouse lines have been developed for cell-type specific gene disruption. But often, the precise tissue-patterns of Cre activity remain incompletely characterized. Two widely used transgenes for conditional gene recombination in hematopoietic cells are Vav-iCre driven from the murine Vav1 promotor, and hCD2-iCre driven from the human CD2 promotor. Vav-iCre expresses active Cre in fetal and adult hematopoietic stem cells and all descendants, hCD2-iCre in immature and mature B and T lymphocytes. To better characterize which hematopoietic cells contain hCD2-iCre activity, we compared EYFP fluorescence in hCD2-iCre+/- R26-stop-EYFP+/- and Vav-iCre+/- R26-stop-EYFP+/-mice. R26-stop-EYFP ubiquitously encodes EYFP preceded by a floxed stop cassette. By removing it, Cre activity induces measurable EYFP expression. Our results confirm the known activity patterns for both Cre transgenes and unveil additional hCD2-iCre mediated reporter gene recombination in common lymphoid progenitors, in natural killer cells and their progenitors, and in plasmacytoid and conventional dendritic cells. This supports previously proposed common lymphoid origins for natural killer cells and subsets of dendritic cells, and indicates the need to consider pleiotropic effects when studying hCD2-iCre mediated conditional knockout mice. Vav-iCre+/- R26-stop-EYFP+/-mice did not show the non-hematopoietic recombination in vascular endothelial cells seen in other Vav-Cre mouse lines, but displayed an unexpected Vav-iCre mediated recombination in a bone cell subset lacking hematopoietic markers. This pinpoints the need to consider stromal cell contributions to phenotypes of Vav-iCre mediated conditional knockout mice. Altogether, our data provide the first detailed assessment of hCD2-iCre and Vav-iCre mediated deletion of floxed genes during lymphocyte development from hematopoietic stem cells and open up novel applications for either Cre-transgenic mouse line.

No MeSH data available.


Related in: MedlinePlus

Murine CD2 mRNA tissue expression profiles.Shown are ImmGen Consortium [53] (www.immgen.org) probe set 10500677 (A-F) and BioGPS [54,55] (www.biogps.org) probe set 1418770_at (G)murine CD2 mRNA expression profiles across (A, B) key hematopoietic cell populations, (C) HSC and HPC populations, (D) B cell developmental and mature populations, (E) T cell and NKT cell developmental and mature populations, (F) DC subsets and (G) multiple hematopoietic tissues and cell types. In (G), non-hematopoietic tissues are not shown because they did not express CD2. HSC, hematopoietic stem cells; HPC, hematopoietic progenitor cells; MPP, multipotent progenitors; MDP, monocyte-DC precursors; CDP, common DC precursors; CLP, common lymphoid progenitors; Fr. A, fraction A pre-pro B cells; pro B, pro B cells; Fr. B,C, fraction B and C pro- and early pre B cells; pre B (D), fraction D late pre B cells; Fr. F, fraction F recirculating mature B cells [19,34]; NK, NK cells; Thy, thymocyte; ETP, early thymocyte progenitor; DN3A, DN3B, DN4, CD4-CD8- thymocyte subsets; DP, CD4+CD8+ thymocytes; SP, CD4+ and CD8+ thymocytes; Treg cells, regulatory T cells; transit. B, transitional B cells.
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pone.0124661.g001: Murine CD2 mRNA tissue expression profiles.Shown are ImmGen Consortium [53] (www.immgen.org) probe set 10500677 (A-F) and BioGPS [54,55] (www.biogps.org) probe set 1418770_at (G)murine CD2 mRNA expression profiles across (A, B) key hematopoietic cell populations, (C) HSC and HPC populations, (D) B cell developmental and mature populations, (E) T cell and NKT cell developmental and mature populations, (F) DC subsets and (G) multiple hematopoietic tissues and cell types. In (G), non-hematopoietic tissues are not shown because they did not express CD2. HSC, hematopoietic stem cells; HPC, hematopoietic progenitor cells; MPP, multipotent progenitors; MDP, monocyte-DC precursors; CDP, common DC precursors; CLP, common lymphoid progenitors; Fr. A, fraction A pre-pro B cells; pro B, pro B cells; Fr. B,C, fraction B and C pro- and early pre B cells; pre B (D), fraction D late pre B cells; Fr. F, fraction F recirculating mature B cells [19,34]; NK, NK cells; Thy, thymocyte; ETP, early thymocyte progenitor; DN3A, DN3B, DN4, CD4-CD8- thymocyte subsets; DP, CD4+CD8+ thymocytes; SP, CD4+ and CD8+ thymocytes; Treg cells, regulatory T cells; transit. B, transitional B cells.

Mentions: We used flow cytometry to analyze EYFP expression as a measure of Cre activity in splenic mature leukocytes from hCD2-iCre+/-R26-stop-EYFP+/- versus R26-stop-EYFP+/- mice. Consistent with the initial characterization of the hCD2-iCre mice and primarily lymphoid CD2 expression [3] (Fig 1), hCD2-iCre mediated gene deletion had occurred in essentially all mature T and B cells (>96% EYFP+ cells, Fig 2). In contrast, macrophages and granulocytes showed negligible hCD2-iCre activity (<5% EYFP+ cells).


hCD2-iCre and Vav-iCre mediated gene recombination patterns in murine hematopoietic cells.

Siegemund S, Shepherd J, Xiao C, Sauer K - PLoS ONE (2015)

Murine CD2 mRNA tissue expression profiles.Shown are ImmGen Consortium [53] (www.immgen.org) probe set 10500677 (A-F) and BioGPS [54,55] (www.biogps.org) probe set 1418770_at (G)murine CD2 mRNA expression profiles across (A, B) key hematopoietic cell populations, (C) HSC and HPC populations, (D) B cell developmental and mature populations, (E) T cell and NKT cell developmental and mature populations, (F) DC subsets and (G) multiple hematopoietic tissues and cell types. In (G), non-hematopoietic tissues are not shown because they did not express CD2. HSC, hematopoietic stem cells; HPC, hematopoietic progenitor cells; MPP, multipotent progenitors; MDP, monocyte-DC precursors; CDP, common DC precursors; CLP, common lymphoid progenitors; Fr. A, fraction A pre-pro B cells; pro B, pro B cells; Fr. B,C, fraction B and C pro- and early pre B cells; pre B (D), fraction D late pre B cells; Fr. F, fraction F recirculating mature B cells [19,34]; NK, NK cells; Thy, thymocyte; ETP, early thymocyte progenitor; DN3A, DN3B, DN4, CD4-CD8- thymocyte subsets; DP, CD4+CD8+ thymocytes; SP, CD4+ and CD8+ thymocytes; Treg cells, regulatory T cells; transit. B, transitional B cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401753&req=5

pone.0124661.g001: Murine CD2 mRNA tissue expression profiles.Shown are ImmGen Consortium [53] (www.immgen.org) probe set 10500677 (A-F) and BioGPS [54,55] (www.biogps.org) probe set 1418770_at (G)murine CD2 mRNA expression profiles across (A, B) key hematopoietic cell populations, (C) HSC and HPC populations, (D) B cell developmental and mature populations, (E) T cell and NKT cell developmental and mature populations, (F) DC subsets and (G) multiple hematopoietic tissues and cell types. In (G), non-hematopoietic tissues are not shown because they did not express CD2. HSC, hematopoietic stem cells; HPC, hematopoietic progenitor cells; MPP, multipotent progenitors; MDP, monocyte-DC precursors; CDP, common DC precursors; CLP, common lymphoid progenitors; Fr. A, fraction A pre-pro B cells; pro B, pro B cells; Fr. B,C, fraction B and C pro- and early pre B cells; pre B (D), fraction D late pre B cells; Fr. F, fraction F recirculating mature B cells [19,34]; NK, NK cells; Thy, thymocyte; ETP, early thymocyte progenitor; DN3A, DN3B, DN4, CD4-CD8- thymocyte subsets; DP, CD4+CD8+ thymocytes; SP, CD4+ and CD8+ thymocytes; Treg cells, regulatory T cells; transit. B, transitional B cells.
Mentions: We used flow cytometry to analyze EYFP expression as a measure of Cre activity in splenic mature leukocytes from hCD2-iCre+/-R26-stop-EYFP+/- versus R26-stop-EYFP+/- mice. Consistent with the initial characterization of the hCD2-iCre mice and primarily lymphoid CD2 expression [3] (Fig 1), hCD2-iCre mediated gene deletion had occurred in essentially all mature T and B cells (>96% EYFP+ cells, Fig 2). In contrast, macrophages and granulocytes showed negligible hCD2-iCre activity (<5% EYFP+ cells).

Bottom Line: R26-stop-EYFP ubiquitously encodes EYFP preceded by a floxed stop cassette.By removing it, Cre activity induces measurable EYFP expression.Our results confirm the known activity patterns for both Cre transgenes and unveil additional hCD2-iCre mediated reporter gene recombination in common lymphoid progenitors, in natural killer cells and their progenitors, and in plasmacytoid and conventional dendritic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
Cre-recombinase mediated conditional deletion of Lox-P site flanked ("floxed") genes is widely used for functional gene annotation in mice. Many different Cre-transgenic mouse lines have been developed for cell-type specific gene disruption. But often, the precise tissue-patterns of Cre activity remain incompletely characterized. Two widely used transgenes for conditional gene recombination in hematopoietic cells are Vav-iCre driven from the murine Vav1 promotor, and hCD2-iCre driven from the human CD2 promotor. Vav-iCre expresses active Cre in fetal and adult hematopoietic stem cells and all descendants, hCD2-iCre in immature and mature B and T lymphocytes. To better characterize which hematopoietic cells contain hCD2-iCre activity, we compared EYFP fluorescence in hCD2-iCre+/- R26-stop-EYFP+/- and Vav-iCre+/- R26-stop-EYFP+/-mice. R26-stop-EYFP ubiquitously encodes EYFP preceded by a floxed stop cassette. By removing it, Cre activity induces measurable EYFP expression. Our results confirm the known activity patterns for both Cre transgenes and unveil additional hCD2-iCre mediated reporter gene recombination in common lymphoid progenitors, in natural killer cells and their progenitors, and in plasmacytoid and conventional dendritic cells. This supports previously proposed common lymphoid origins for natural killer cells and subsets of dendritic cells, and indicates the need to consider pleiotropic effects when studying hCD2-iCre mediated conditional knockout mice. Vav-iCre+/- R26-stop-EYFP+/-mice did not show the non-hematopoietic recombination in vascular endothelial cells seen in other Vav-Cre mouse lines, but displayed an unexpected Vav-iCre mediated recombination in a bone cell subset lacking hematopoietic markers. This pinpoints the need to consider stromal cell contributions to phenotypes of Vav-iCre mediated conditional knockout mice. Altogether, our data provide the first detailed assessment of hCD2-iCre and Vav-iCre mediated deletion of floxed genes during lymphocyte development from hematopoietic stem cells and open up novel applications for either Cre-transgenic mouse line.

No MeSH data available.


Related in: MedlinePlus