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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

hCD2-iCre activity in T, B and NK cell development.(A) Thymocyte populations were identified by the gating strategy in the upper panel [13,31–33]. Lower panel, EYFP expression in the indicated thymocyte populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (B) Upper panel, subsets of developing B cells in the BM were distinguished as in [19]. Lower panel, EYFP expression in the indicated BM B cell populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (C) Upper panels, NK cell progenitors (NKP), immature (iNK) and mature (mNK) NK cells were identified using the indicated gating strategy [18,29]. Lower panels, EYFP expression in the indicated BM and splenic NK cell populations from (top, n = 3 per genotype) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or (bottom, n = 2 per genotype) from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers indicate % EYFP+ cells in the respective Cre+/- mice. Representative of three independent experiments with hCD2-iCre transgenic mice, and of two independent experiments with Vav-iCre transgenic mice.
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pone.0124661.g003: hCD2-iCre activity in T, B and NK cell development.(A) Thymocyte populations were identified by the gating strategy in the upper panel [13,31–33]. Lower panel, EYFP expression in the indicated thymocyte populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (B) Upper panel, subsets of developing B cells in the BM were distinguished as in [19]. Lower panel, EYFP expression in the indicated BM B cell populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (C) Upper panels, NK cell progenitors (NKP), immature (iNK) and mature (mNK) NK cells were identified using the indicated gating strategy [18,29]. Lower panels, EYFP expression in the indicated BM and splenic NK cell populations from (top, n = 3 per genotype) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or (bottom, n = 2 per genotype) from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers indicate % EYFP+ cells in the respective Cre+/- mice. Representative of three independent experiments with hCD2-iCre transgenic mice, and of two independent experiments with Vav-iCre transgenic mice.

Mentions: Lymphoid NKT cells develop in the thymus from CD4+CD8+ precursors and have similar signaling requirements as developing T cells [28]. Both NKT precursors and NKT cells express CD2 mRNA (Fig 1B and 1E). CD4+CD8+ thymocytes have high hCD2-iCre activity (Fig 3). Consistent with these findings, we found >83% EYFP+ NKT cells (Fig 2).


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

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

hCD2-iCre activity in T, B and NK cell development.(A) Thymocyte populations were identified by the gating strategy in the upper panel [13,31–33]. Lower panel, EYFP expression in the indicated thymocyte populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (B) Upper panel, subsets of developing B cells in the BM were distinguished as in [19]. Lower panel, EYFP expression in the indicated BM B cell populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (C) Upper panels, NK cell progenitors (NKP), immature (iNK) and mature (mNK) NK cells were identified using the indicated gating strategy [18,29]. Lower panels, EYFP expression in the indicated BM and splenic NK cell populations from (top, n = 3 per genotype) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or (bottom, n = 2 per genotype) from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers indicate % EYFP+ cells in the respective Cre+/- mice. Representative of three independent experiments with hCD2-iCre transgenic mice, and of two independent experiments with Vav-iCre transgenic mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124661.g003: hCD2-iCre activity in T, B and NK cell development.(A) Thymocyte populations were identified by the gating strategy in the upper panel [13,31–33]. Lower panel, EYFP expression in the indicated thymocyte populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (B) Upper panel, subsets of developing B cells in the BM were distinguished as in [19]. Lower panel, EYFP expression in the indicated BM B cell populations from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers denote % EYFP+ cells within the indicated population of hCD2-iCre+/-R26-stop-EYFP+/- mice. Representative of three independent experiments (n = 3). (C) Upper panels, NK cell progenitors (NKP), immature (iNK) and mature (mNK) NK cells were identified using the indicated gating strategy [18,29]. Lower panels, EYFP expression in the indicated BM and splenic NK cell populations from (top, n = 3 per genotype) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or (bottom, n = 2 per genotype) from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Numbers indicate % EYFP+ cells in the respective Cre+/- mice. Representative of three independent experiments with hCD2-iCre transgenic mice, and of two independent experiments with Vav-iCre transgenic mice.
Mentions: Lymphoid NKT cells develop in the thymus from CD4+CD8+ precursors and have similar signaling requirements as developing T cells [28]. Both NKT precursors and NKT cells express CD2 mRNA (Fig 1B and 1E). CD4+CD8+ thymocytes have high hCD2-iCre activity (Fig 3). Consistent with these findings, we found >83% EYFP+ NKT cells (Fig 2).

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