Basic Information

Common Name
B-hTSLP/hTSLPR/hIL7R mice
Background
C57BL/6
Catalog Number
111951

Gene targeting strategy

Gene targeting strategy for B-hTSLP/hTSLPR/hIL7R mice. Exons 1-5 of the mouse Tslp gene that encodes the full-length protein were replaced by human TSLP exons 1-4 in B-hTSLP/hTSLPR mice. The signal peptide, extracellular and transmembrane region of human TSLPR gene and the cytoplasmic region of mouse Tslpr gene were constructed into a chimeric CDS vector and inserted into the mouse exon 2. The targeted mice will express the chimeric TSLPR protein, while mouse TSLPR will no longer express in B-hTSLP/hTSLPR mice. The exons 1~6 of mouse Il7r gene that encode the extracellular region were replaced by human IL7R exons 1~6 in B-hIL7R mice. B-hTSLP/hTSLPR mice and B-hIL7R mice were mated together to obtain trigeneic humanized B-hTSLP/hTSLPR/hIL7R mice.

Protein expression analysis

Species-specific TSLPR protein expression analysis in wild-type and humanized mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL7R (H/H), B-hTSLP/hTSLPR (H/H) and B-hTSLP/hTSLPR/hIL7R (H/H) mice, and analyzed by flow cytometry using species-specific anti-TSLPR antibodies. Mouse TSLPR protein was detected on cDCs, pDCs and non DCs from wild-type and B-hIL7R mice, while human TSLPR protein was detected on cDCs, pDCs and non DCs from B-hTSLP/hTSLPR and B-hTSLP/hTSLPR/hIL7R mice.

Species-specific IL7R protein expression analysis in wild-type and humanized mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL7R (H/H), B-hTSLP/hTSLPR (H/H) and B-hTSLP/hTSLPR/hIL7R (H/H) mice, and analyzed by flow cytometry using species-specific anti-IL7R antibodies. Mouse IL7R protein was detected on non DCs from wild-type C57BL/6 and B-hTSLP/hTSLPR mice, but spleen-derived DCs barely expressed mouse IL7R. Human IL7R protein was detected on non DCs from B-hIL7R and B-hTSLP/hTSLPR/hIL7R mice, but spleen-derived DCs barely expressed human IL7R.

Human TSLPR and IL7R expression analysis in cDC1 from bone marrow. Bone marrow were collected from wild type C57BL/6 mice, homozygous B-hIL7R mice, B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR/hIL7R mice. Human TSLPR and IL7R expressed on cDC1 were analyzed by flow cytometry with species-specific antibodies. Human TSLPR was highly expressed on the cDC1 in B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR/hIL7R mice. However, Human IL7R was barely expressed on the cDC1 in B-hIL7R mice and B-hTSLP/hTSLPR/hIL7R mice.

Functional analysis

TARC induction with species-specific TSLP protein in wild-type and humanized mice. Dendritic cells were respectively induced with FLT3L from bone marrow of the four strains, and stimulated with human TSLP or mouse TSLP in vitro. Concentration of mouse TARC secreted from DCs was analyzed by ELISA. Mouse TARC was successfully induced with human TSLP, but not mouse TSLP in homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR/hIL7R mice. The level of mTARC in B-hTSLP/hTSLPR/hIL7R mice was higher than that in B-hTSLP/hTSLPR mice. Meanwhile mouse TARC was successfully induced with mouse TSLP, but not human TSLP in wild-type C57BL/6 mice and B-hIL7R mice. The level of mTARC in wild-type C57BL/6 mice was higher than that in B-hIL7R mice. Results indicated that TSLP and TSLPR are not cross-reactive between mouse and human. Human TSLP can activate the dendritic cells of B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR/hIL7R mice. IL-7R contributed to the production of mTARC in TSLP/TSLPR/IL7R humanized mice and wild type mice induced by corresponding TSLP.