Basic Information

Strain Name
C57BL/6-Adora2atm1(ADORA2A)/Bcgen
Stock Number
110120
Common Name
B-hA2AR mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
A2AR (ADORA2A, adenosine A2A receptor)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

A2AR encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily, which is subdivided into classes and subtypes. The receptors are seven-pass transmembrane proteins that respond to extracellular cues and activate intracellular signal transduction pathways. This protein, an adenosine receptor of A2A subtype, uses adenosine as the preferred endogenous agonist and preferentially interacts with the G(s) and G(olf) family of G proteins to increase intracellular cAMP levels. It plays an important role in many biological functions, such as cardiac rhythm and circulation, cerebral and renal blood flow, immune function, pain regulation, and sleep. It has been implicated in pathophysiological conditions such as inflammatory diseases and neurodegenerative disorders.

Details

mRNA expression analysis

Strain specific analysis of A2AR gene expression in WT and hA2AR mice by RT-PCR. Mouse A2ar mRNA was detectable only in splenocytes of wild-type (+/+) mice. Human A2AR mRNA was detectable only in H/H, but not in +/+ mice.

Protein expression analysis in T cells

Strain specific A2AR expression analysis in homozygous B-hA2AR mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hA2AR (H/H) mice stimulated with anti-CD3ε (7.5 μg/mice, stimulation for 24 hours, i.p.) in vivo, and analyzed by flow cytometry with species-specific anti-A2AR antibody. Mouse A2AR was detectable in WT mice, human A2AR was detectable in homozygous B-hA2AR due to the anti-mouse A2AR antibody also cross-reacts with human A2AR.

Protein expression analysis in CD4+ T cells

Strain specific A2AR expression analysis in homozygous B-hA2AR mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hA2AR (H/H) mice stimulated with anti-CD3ε (7.5 μg/mice, stimulation for 24 hours, i.p.) in vivo, and analyzed by flow cytometry with species-specific anti-A2AR antibody. Mouse A2AR was detectable in WT mice, human A2AR was detectable in homozygous B-hA2AR due to the anti-mouse A2AR antibody also cross-reacts with human A2AR.

Function analysis

cAMP measurements in spleen and bone of wild-type mice and B-hA2AR mice incubated with different concentration of CGS21680 (an A2AR agonist), and analyzed by LANCE® Ultra cAMP Kit (PerkinElmer).

The cAMP levels of spleen cells and bone marrow cells in B-hA2AR mice were higher than those in C57BL/6 mice at 500μM CGS21680. It was speculated that B-hA2AR mice were more sensitive to the stimulation of CGS201680 than wild mice. At the background level, there was no significant difference in A2AR mediated cAMP pathway between B-hA2AR and C57BL/6 mice. Compared with C57BL/6, B-hA2AR more effectively mediated the A2AR-dependent cAMP pathway under strong stimulation.

Analysis of spleen leukocyte subpopulations in B-hA2AR mice


Analysis of splenic leukocyte subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hA2AR mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocytes/macrophages, and DC were similar in homozygous B-hA2AR mice and C57BL/6 mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of spleen leukocyte subpopulations in B-hA2AR mice


Analysis of splenic T cell subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hA2AR mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on TCRβ+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hA2AR and C57BL/6 mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations in B-hA2AR mice


Analysis of blood leukocyte subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hA2AR mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocytes/macrophages, and DC were similar in homozygous B-hA2AR mice and C57BL/6 mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations in B-hA2AR mice


Analysis of blood T cell subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hA2AR mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on TCRβ+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hA2AR and C57BL/6 mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.