Limits...
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 HSC and HPC subsets.(A) Upper panels, HPC subsets were identified using the indicated gating strategy [24,25]. Lower panels, EYFP expression in CLP (Lin-Flk2+IL-7Rα+CD27+), MEP (Lin-IL-7Rα-c-kit+CD16/32-CD34-), CMP (Lin-IL-7Rα-c-kit+CD16/32-/loCD34+) and GMP (Lin-IL-7Rα-c-kit+CD16/32+CD34+) from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Representative of three independent experiments. (B) Upper panels, phenotypic HSC/MPP subsets were identified using the indicated gating strategy [23,35,56]. Lower panels, EYFP expression in phenotypic LT-HSC (Lin-c-kit+Sca-1+CD150+CD34-CD48-Flk2-), ST-HSC1 (Lin-c-kit+Sca-1+CD150+CD34+CD48-Flk2-), ST-HSC2 (Lin-c-kit+Sca-1+CD150+CD34+CD48+Flk2-), ST-HSC3 (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2-) and MPP (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2+) from the mice in (A). Representative of at least two independent experiments. Numbers indicate % EYFP+ cells in the respective Cre+/- mice (n = 3 for experiments with hCD2-iCre transgenic mice, n = 2 for experiments with Vav-iCre transgenic mice).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401753&req=5

pone.0124661.g004: hCD2-iCre activity in HSC and HPC subsets.(A) Upper panels, HPC subsets were identified using the indicated gating strategy [24,25]. Lower panels, EYFP expression in CLP (Lin-Flk2+IL-7Rα+CD27+), MEP (Lin-IL-7Rα-c-kit+CD16/32-CD34-), CMP (Lin-IL-7Rα-c-kit+CD16/32-/loCD34+) and GMP (Lin-IL-7Rα-c-kit+CD16/32+CD34+) from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Representative of three independent experiments. (B) Upper panels, phenotypic HSC/MPP subsets were identified using the indicated gating strategy [23,35,56]. Lower panels, EYFP expression in phenotypic LT-HSC (Lin-c-kit+Sca-1+CD150+CD34-CD48-Flk2-), ST-HSC1 (Lin-c-kit+Sca-1+CD150+CD34+CD48-Flk2-), ST-HSC2 (Lin-c-kit+Sca-1+CD150+CD34+CD48+Flk2-), ST-HSC3 (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2-) and MPP (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2+) from the mice in (A). Representative of at least two independent experiments. Numbers indicate % EYFP+ cells in the respective Cre+/- mice (n = 3 for experiments with hCD2-iCre transgenic mice, n = 2 for experiments with Vav-iCre transgenic mice).

Mentions: Innate NK cells are commonly thought to develop from CLP via Lin-CD3-CD122+NK1.1+ NK cell progenitors (NKP) [14,18,29]. However, at least in vitro, human myeloid progenitors can also produce NK cells [30]. We found ≥36% EYFP+ mature NK1.1+CD11b+ splenic NK cells (mNK), ≥49% EYFP+ immature NK cells (iNK), ≥55% EYFP+ NK cell progenitors (NKP, Figs 2 and 3C) and ≥27% EYFP+ CLP (Fig 4A) in hCD2-iCre+/-R26-stop-EYFP+/- mice. This is consistent with CD2 mRNA expression in NK cells (Fig 1A,1B and 1G) and supports derivation of many NKP and NK cells from CLP.


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 HSC and HPC subsets.(A) Upper panels, HPC subsets were identified using the indicated gating strategy [24,25]. Lower panels, EYFP expression in CLP (Lin-Flk2+IL-7Rα+CD27+), MEP (Lin-IL-7Rα-c-kit+CD16/32-CD34-), CMP (Lin-IL-7Rα-c-kit+CD16/32-/loCD34+) and GMP (Lin-IL-7Rα-c-kit+CD16/32+CD34+) from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Representative of three independent experiments. (B) Upper panels, phenotypic HSC/MPP subsets were identified using the indicated gating strategy [23,35,56]. Lower panels, EYFP expression in phenotypic LT-HSC (Lin-c-kit+Sca-1+CD150+CD34-CD48-Flk2-), ST-HSC1 (Lin-c-kit+Sca-1+CD150+CD34+CD48-Flk2-), ST-HSC2 (Lin-c-kit+Sca-1+CD150+CD34+CD48+Flk2-), ST-HSC3 (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2-) and MPP (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2+) from the mice in (A). Representative of at least two independent experiments. Numbers indicate % EYFP+ cells in the respective Cre+/- mice (n = 3 for experiments with hCD2-iCre transgenic mice, n = 2 for experiments with Vav-iCre transgenic mice).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124661.g004: hCD2-iCre activity in HSC and HPC subsets.(A) Upper panels, HPC subsets were identified using the indicated gating strategy [24,25]. Lower panels, EYFP expression in CLP (Lin-Flk2+IL-7Rα+CD27+), MEP (Lin-IL-7Rα-c-kit+CD16/32-CD34-), CMP (Lin-IL-7Rα-c-kit+CD16/32-/loCD34+) and GMP (Lin-IL-7Rα-c-kit+CD16/32+CD34+) from hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from Vav-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms). Representative of three independent experiments. (B) Upper panels, phenotypic HSC/MPP subsets were identified using the indicated gating strategy [23,35,56]. Lower panels, EYFP expression in phenotypic LT-HSC (Lin-c-kit+Sca-1+CD150+CD34-CD48-Flk2-), ST-HSC1 (Lin-c-kit+Sca-1+CD150+CD34+CD48-Flk2-), ST-HSC2 (Lin-c-kit+Sca-1+CD150+CD34+CD48+Flk2-), ST-HSC3 (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2-) and MPP (Lin-c-kit+Sca-1+CD150-CD34+CD48+Flk2+) from the mice in (A). Representative of at least two independent experiments. Numbers indicate % EYFP+ cells in the respective Cre+/- mice (n = 3 for experiments with hCD2-iCre transgenic mice, n = 2 for experiments with Vav-iCre transgenic mice).
Mentions: Innate NK cells are commonly thought to develop from CLP via Lin-CD3-CD122+NK1.1+ NK cell progenitors (NKP) [14,18,29]. However, at least in vitro, human myeloid progenitors can also produce NK cells [30]. We found ≥36% EYFP+ mature NK1.1+CD11b+ splenic NK cells (mNK), ≥49% EYFP+ immature NK cells (iNK), ≥55% EYFP+ NK cell progenitors (NKP, Figs 2 and 3C) and ≥27% EYFP+ CLP (Fig 4A) in hCD2-iCre+/-R26-stop-EYFP+/- mice. This is consistent with CD2 mRNA expression in NK cells (Fig 1A,1B and 1G) and supports derivation of many NKP and NK cells from CLP.

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