Basic Information

Strain Name
C57BL/6-Lrrc32tm1(LRRC32)/Bcgen
Stock Number
110102
Common Name
B-hGARP mice
Background
C57BL/6
NCBI Gene ID
Related Genes
LRRC32 (Leucine-Rich Repeat-Containing Protein 32), GARP (Glycoprotein-A repetitions predominant)

Targeting Strategy

Exon 1~2 of mouse Lrrc32 gene, which encodes the extracellular domain, was replaced by human LRRC32 exon 1~2 in B-hGARP mice.

mRNA expression analysis

Species-specific analysis of GARP gene expression in WT and B-hGARP mice by RT-PCR. Mouse Garp mRNA was detectable in splenocytes of wild-type (+/+) mice. Human GARP mRNA was detectable only in H/H, but not in +/+ mice.

Protein expression analysis

Protein expression analysis in Treg cells

Strain specific GARP expression analysis in homozygous B-hGARP mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hGARP (H/H) mice, and analyzed by flow cytometry with species-specific GARP antibody. Mouse GARP was detectable in WT mice. Human GARP was exclusively detectable in homozygous B-hGARP but not WT mice.

Analysis of spleen leukocyte subpopulations in B-hGARP mice

Analysis of splenic leukocyte subpopulations by FACS.Splenocytes were isolated from female C57BL/6 and B-hGARP 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, monocyte/macrophages, and DC were similar in homozygous B-hGARP mice and C57BL/6 mice, demonstrating that introduction of hGARP in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in spleen. Similar results were obtained for blood leukocyte analysis. Values are expressed as mean ± SEM.


Analysis of splenic T cell subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hGARP mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on CD3+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hGARP and C57BL/6 mice, demonstrating that introduction of hGARP 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-hGARP mice



Analysis of blood leukocyte subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hGARP 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, monocyte/macrophages, and DC were similar in homozygous B-hGARP mice and C57BL/6 mice, demonstrating that introduction of hGARP 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 T cell subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hGARP mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on CD3+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hGARP and C57BL/6 mice, demonstrating that introduction of hGARP 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.

Functional analysis

Combination therapy of anti-mouse PD-1 antibody and anti-human GARP/latent-TGFβ1 antibody in humanized B-hGARP mice

Anti-tumor activity of anti-mouse PD-1 antibody combined with anti-human GARP/latent-TGFβ1 antibody in humanized B-hGARP mice. Murine colon cancer cells were subcutaneously implanted into homozygous B-hGARP mice (female, 7-week-old, n=6). Mice were grouped when tumor volume reached approximately 50~70 mm3, at which time they were treated with anti-mouse PD-1 antibody and/or anti-human GARP/latent-TGFβ1 antibody with doses and schedules indicated in panel A. (A) Anti-human GARP/latent-TGFβ1 (ABBV151, in house) antibody combined with anti-mouse PD-1 antibody drastically inhibited MC38 tumor growth in B-hGARP mice, (B) without negatively impacting body weight. B-hGARP mice provide a powerful preclinical model for in vivo evaluation of anti-human GARP antibodies. Values are expressed as mean ± SEM.

Poster

AACR 2022: Generation and Validation of Humanized GARP Mice for Testing Novel Anti-Human GARP Antibodies