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Comparative Study
. 2014 Jun:408:89-100.
doi: 10.1016/j.jim.2014.05.009. Epub 2014 May 22.

Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice

Clare L Abram  1 , Gray L Roberge  1 , Yongmei Hu  1 , Clifford A Lowell  2
Affiliations
Comparative Study

Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice

Clare L Abram et al. J Immunol Methods. 2014 Jun.

Abstract

Since the first example of conditional gene targeting in mice in 1994, the use of Cre recombinase and loxP flanked sequences has become an invaluable technique to generate tissue and temporal specific gene knockouts. The number of mouse strains expressing floxed-gene sequences, and tissue-specific or temporal-specific Cre-recombinase that have been reported in the literature has grown exponentially. However, increased use of this technology has highlighted several problems that can impact the interpretation of any phenotype observed in these mouse models VSports手机版. In particular, accurate knowledge of the specificity of Cre expression in each strain is critical in order to make conclusions about the role of specific cell types in the phenotypes observed. Cre-mediated deletion specificity and efficiency have been described in many different ways in the literature, making direct comparisons between these Cre strains impossible. Here we report crossing thirteen different myeloid-Cre mouse strains to ROSA-EYFP reporter mice and assaying YFP expression in a variety of naïve unstimulated hematopoietic cells, in parallel. By focusing on myeloid subsets, we directly compare the relative efficiency and specificity of myeloid deletion in these strains under steady-state conditions. .

Keywords: Cre-recombinase; Dendritic cell; Floxed; Macrophage; Neutrophil V体育安卓版. .

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Figure 1
Figure 1
Gating strategy of myeloid cell types analyzed by flow cytometry. DAPI –ve autofluorescence low cells were gated as indicated for all populations, except for macrophages, where the autofluorescent population was included and representative FACS plots are shown. (A) In peripheral blood, T cells (TCRβ+; 18.7±2.1%), B cells (CD19+; 32.4±3.7%), neutrophils (CD11b+Gr1hi; 21.6±6.4%) and monocytes (CD11b+F4/80+; 2.16±1.2%) were examined. Monocytes were further subdivided as inflammatory (Ly6chi7/4hi; 2.4±0.6%) or resident (Ly6clo7/4lo; 3.3±0.4%). Peripheral blood percentages refer to the percent of total leukocytes. (B) Alveolar macrophages were assayed from the bronchio-alveolar lavage (BAL) and defined as CD11chiF4/80hi (94±4.2%). (C) In the bone marrow, T cells (0.9±0.14%), B cells (6.3±1.0%) and neutrophils (55.2±1.9%) were defined as for peripheral blood. (D) In the peritoneum, peritoneal macrophages (CD45+F4/80+; 58.9±7.2%), mast cells (CD45+c-Kit+; 2.2±0.4%), B1 B cells (CD19+CD11bint; 8.5±2.5%) and B2 B cells (CD19+CD11b−; 13.9±4.9%) were assayed. (E) In the spleen, CD11b+ cells accounted for 17.1±2.4% of total splenocytes. T cells (34.8±2.6%), B cells (43.5±1.8%) and neutrophils (3.7±1.1%) were defined as for peripheral blood. Splenic macrophages were defined as marginal zone (MZ, CD11b+F4/80int; 3.9±0.6%) and red pulp (RP, autofluorescencehiF4/80hiCD11b−; 4.4±0.7%). For the following low abundance populations, a B220−TCRβ− gate was analyzed but the percentage of total splenocytes is given. cDCs (CD11chi; 1.37±0.3%), further subdivided as “lymphoid” (CD8+; 0.22±0.06%) or “myeloid” (CD11b+; 0.96±0.2%), pDCs (CD11cintLy6c+B220+; 0.3±0.06%), NK cells (NK1.1+NKp46+; 3.6±1.3%), basophils (CD49b+IgE+; 0.3±0.07%) and eosinophils (SiglecF+CD11b+; 0.9±0.3%) were assayed. Cell frequencies are calculated from the average of 5-6 mice ± StdDev.
Figure 2
Figure 2
YFP+ cells from spleen CD11b+ gate (A). Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM). To illustrate the heterogeneity of deletion seen in the CD11b-cre strain, the percentage of YFP+CD11b+ cells from the peripheral blood, bone marrow and spleen were divided into two groups based on mice with higher or lower %YFP+ cells (B).
Figure 3
Figure 3
YFP+ cells from neutrophil populations. Neutrophils were defined as in Figure 1 and assayed in spleen (A), peripheral blood (B) and bone marrow (C). Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM).
Figure 4
Figure 4
YFP+ cells from peripheral blood monocyte populations. Total blood monocytes (A) were subdivided into inflammatory and resident monocyte populations as defined in Figure 1 (B). Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM). (C) CD41hi platelets from peripheral blood are YFP+ in the PF4-cre strain.
Figure 5
Figure 5
A fraction of peripheral blood leukocytes in the PF4-cre strain appear YFP+ due to platelet adhesion. (A) Cells were examined using the Amnis Imagestream and representative images are shown. (B) YFP+ peritoneal macrophages isolated from the PF4-cre strain do not show staining with the platelet marker, CD41.
Figure 6
Figure 6
YFP+ cells from mature macrophage populations. Tissue macrophage populations from (A) bronchio-alveolar lavage, (B) peritoneum and (C, D) spleen as defined in Figure 1 were assayed. Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM).
Figure 7
Figure 7
YFP+ cells from splenic dendritic cell populations. (A) cDCs and (B) pDCs as defined in Figure 1 were assayed. The cDC population was further subdivided into CD8+ “lymphoid” DCs (C) and CD11b+ “myeloid” DCs (D) to highlight differential Cre expression observed in the CD11c-cre and Cx3cr1-cre strains. Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM).
Figure 8
Figure 8
YFP+ cells from other myeloid cell populations. (A) Peritoneal mast cells, (B) splenic NK cells, (C) splenic basophils and (E) splenic eosinophils as defined in Figure 1 were assayed. Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM). (D) Cre activity in the YFP+ basophils from Basoph8-cre Tg/+ mice was demonstrated by crossing these mice with an Ai14 reporter strain and confirming that these cells were also Ai14+.
Figure 9
Figure 9
YFP+ cells from lymphoid populations. T cells (A, C, E), B cells (B, D, F) and peritoneal B1 and B2 cells (G and H) as defined in Figure 1 were assayed. Bars represent the average of 5-14 mice analyzed per Cre strain (error bars indicate SEM).
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