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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

Vav-iCre activity in non-lymphoid bone cells.(A) Gating strategy. After flushing out the BM, bones were digested with collagenase and remaining cells stained for expression of the indicated markers. (B) EYFP expression on EC (Lin-CD45-CD31+), OB (Lin-CD45-CD31-CD51+Sca-1-), MSC (Lin-CD45-CD31-CD51+Sca-1+) and abundant Lin-CD45-CD31-CD51-Sca-1- bone cells of unknown identity [22] from (upper panels) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from (lower panels) 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 (n = 3).
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pone.0124661.g005: Vav-iCre activity in non-lymphoid bone cells.(A) Gating strategy. After flushing out the BM, bones were digested with collagenase and remaining cells stained for expression of the indicated markers. (B) EYFP expression on EC (Lin-CD45-CD31+), OB (Lin-CD45-CD31-CD51+Sca-1-), MSC (Lin-CD45-CD31-CD51+Sca-1+) and abundant Lin-CD45-CD31-CD51-Sca-1- bone cells of unknown identity [22] from (upper panels) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from (lower panels) 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 (n = 3).

Mentions: Besides hematopoietic cells, older Vav-Cre lines also recombine floxed genes in the testis, and in vascular endothelial cells (EC) or precursors which do not express Vav, possibly due to Cre-transgene or LacZ reporter insertion effects [3,6]. To assess whether this also occurs in our hCD2-iCre and Vav-iCre lines, we next analyzed EYFP expression in stromal and endothelial cells from collagenase-digested, BM depleted bones [22]. We found no EYFP expression in hCD2-iCre+/-R26-stop-EYFP+/- and Vav-iCre+/-R26-stop-EYFP+/- Lin-CD45-CD31+ EC, Lin-CD45-CD31-CD51+Sca-1- osteoblasts (OB) and Lin-CD45-CD31-CD51+Sca-1+ mesenchymal stem cells (MSC, Fig 5). As previously reported [22], most Lin-CD45- cells in collagenase-digested bones were CD31-CD51-Sca-1-. About 30% of these cells were EYFP+, the other 70% EYFPlow in Vav-iCre+/-R26-stop-EYFP+/- but not hCD2-iCre+/-R26-stop-EYFP+/- mice (Fig 5), indicating a specific Vav-iCre activity. These cells are not EC and do not bear surface markers of mast cells (FcεR) or erythroid progenitors (CD41) as assessed by FACS. Their identity requires further investigation. Altogether, these data confirm potent Vav-iCre activity in all HSC and HPC subsets and identify a novel Vav-iCre activity containing Lin-CD45-CD31-CD51-Sca-1- subset of bone 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)

Vav-iCre activity in non-lymphoid bone cells.(A) Gating strategy. After flushing out the BM, bones were digested with collagenase and remaining cells stained for expression of the indicated markers. (B) EYFP expression on EC (Lin-CD45-CD31+), OB (Lin-CD45-CD31-CD51+Sca-1-), MSC (Lin-CD45-CD31-CD51+Sca-1+) and abundant Lin-CD45-CD31-CD51-Sca-1- bone cells of unknown identity [22] from (upper panels) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from (lower panels) 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 (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124661.g005: Vav-iCre activity in non-lymphoid bone cells.(A) Gating strategy. After flushing out the BM, bones were digested with collagenase and remaining cells stained for expression of the indicated markers. (B) EYFP expression on EC (Lin-CD45-CD31+), OB (Lin-CD45-CD31-CD51+Sca-1-), MSC (Lin-CD45-CD31-CD51+Sca-1+) and abundant Lin-CD45-CD31-CD51-Sca-1- bone cells of unknown identity [22] from (upper panels) hCD2-iCre+/-R26-stop-EYFP+/- (open histograms) or R26-stop-EYFP+/- mice (shaded histograms), or from (lower panels) 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 (n = 3).
Mentions: Besides hematopoietic cells, older Vav-Cre lines also recombine floxed genes in the testis, and in vascular endothelial cells (EC) or precursors which do not express Vav, possibly due to Cre-transgene or LacZ reporter insertion effects [3,6]. To assess whether this also occurs in our hCD2-iCre and Vav-iCre lines, we next analyzed EYFP expression in stromal and endothelial cells from collagenase-digested, BM depleted bones [22]. We found no EYFP expression in hCD2-iCre+/-R26-stop-EYFP+/- and Vav-iCre+/-R26-stop-EYFP+/- Lin-CD45-CD31+ EC, Lin-CD45-CD31-CD51+Sca-1- osteoblasts (OB) and Lin-CD45-CD31-CD51+Sca-1+ mesenchymal stem cells (MSC, Fig 5). As previously reported [22], most Lin-CD45- cells in collagenase-digested bones were CD31-CD51-Sca-1-. About 30% of these cells were EYFP+, the other 70% EYFPlow in Vav-iCre+/-R26-stop-EYFP+/- but not hCD2-iCre+/-R26-stop-EYFP+/- mice (Fig 5), indicating a specific Vav-iCre activity. These cells are not EC and do not bear surface markers of mast cells (FcεR) or erythroid progenitors (CD41) as assessed by FACS. Their identity requires further investigation. Altogether, these data confirm potent Vav-iCre activity in all HSC and HPC subsets and identify a novel Vav-iCre activity containing Lin-CD45-CD31-CD51-Sca-1- subset of bone 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