Basic Information
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Targeting strategy
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Gene targeting strategy for B-NDG hIL15, FcγR KO mice. The CDS region of the human IL15 gene was inserted after the 5′UTR of the mouse Il15 gene in B-NDG hIL15 mice. Then the full sequences of mouse gene Fcgr1 were knocked out while the full genomic sequences from Fcgr2b to Fcgr3 were also knocked out in B-NDG hIL15, FcγR KO mice.
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Protein expression analysis
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Strain specific FcγRs expression analysis in homozygous B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice by flow cytometry. Spleens were collected from homozygous B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice, and analyzed by flow cytometry with four species-specific anti-FcγR antibodies. Mouse FcγRIIb and FcγRIV were detectable in B-NDG hIL15 mice but not in B-NDG hIL15, FcγR KO mice. Surprisingly, Mouse FcγRI and FcγRIII were not detectable in B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice (Data was not shown). So we arranged an RT-PCR experiment next.
Strain specific IL15 expression analysis in B-NDG mice, homozygous B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice by ELISA. Serum was collected from male B-NDG mice (9-week-old) , homozygous B-NDG hIL15 mice (9-week-old) and B-NDG hIL15, FcγR KO mice (12-week-old), and analyzed by ELISA with species-specific IL15 antibody. Human IL15 was detectable in homozygous B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice but not in B-NDG mice. The expression level in B-NDG hIL15, FcγR KO mice was similar to that in B-NDG hIL15 mice.
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Analysis of leukocyte subpopulations in blood
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Analysis of blood leukocyte subpopulations by flow cytometry. Blood were isolated from male B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice (n=3). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of granulocytes, monocytes, macrophages and DCs in homozygous B-NDG hIL15, FcγR KO mice were similar to those in the B-NDG hIL15 mice, demonstrating that knock out of FcγRs does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
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Analysis of leukocytes cell subpopulation in spleen
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Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from male B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice (n=3). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of granulocytes, monocytes, macrophages and DCs in homozygous B-NDG hIL15, FcγR KO mice were similar to those in the B-NDG hIL15 mice, demonstrating that knock out of FcγRs does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
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Summary
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Protein expression analysis:
- Mouse FcγRI, FcγRIIb, FcγRIII and FcγRIV were detectable in B-NDG hIL15 mice but not in B-NDG hIL15, FcγR KO mice.
- Human IL15 was detectable in homozygous B-NDG hIL15 mice and B-NDG hIL15, FcγR KO mice but not in B-NDG mice. The expression level in B-NDG hIL15, FcγR KO mice was similar to that in B-NDG hIL15 mice.
- Leukocytes subpopulation analysis：
Knock out of FcγRs does not change the overall development, differentiation or distribution of these cell types in blood and spleen.
