• Fms-like tyrosine kinase receptor 3 (Flt3) is a membrane protein that is strongly expressed on hematopoietic stem cells (HSCs). FLT3LG (FLT3 ligand) is a growth factor that can stimulate the proliferation, differentiation, and survival of these cells when it binds to its receptor FLT3. The Flt3/Flt3L signaling axis indeed plays an important role in the production of conventional dendritic cells (cdc) and plasmacytoid dendritic cells (pDCs). Both types of dendritic cells are critical members of the immune system involved in processes such as fighting infections and regulating immune responses.
• Gene editing strategy: The exons 2-9 of mouse Flt3 gene are replaced by the reverse inserted exons 1-9 of human FLT3LG including promoter, 5’UTR and 3’UTR region in B-NDG hFLT3LG, Flt3 KO mice.
• Protein expression analysis: Human FLT3LG was detectable in heterozygous B-NDG hFLT3LG, Flt3 KO mice but not in B-NDG mice.
• Application: This product results in a high level of immune system reconstitution after the transplantation of artificial hematopoietic stem cells, especially for DC cells.

Strain specific analysis of mFlt3 and hFLT3LG mRNA expression in B-NDG mice and homozygous B-NDG hFLT3LG, Flt3 KO mice by RT-PCR. Bone marrow RNA were isolated from B-NDG mice and homozygous B-NDG hFLT3LG, Flt3 KO mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Flt3 primers and human FLT3LG primers. Mouse Flt3 mRNA was detectable only in B-NDG mice but not in homozygous B-NDG hFLT3LG, Flt3 KO mice. Human FLT3LG mRNA was detectable only in homozygous B-NDG hFLT3LG, Flt3 KO mice.

Strain specific FLT3 expression analysis in B-NDG hFLT3LG, Flt3 KO mice by flow cytometry. Bone marrow was collected from B-NDG mice and B-NDG hFLT3LG, Flt3 KO mice (H/H). Protein expression was analyzed with species-specific anti-mouse FLT3 antibody (Biolegend, 135309). mFLT3 was detectable in DCs and CD117+ cells of B-NDG mice, but not in homozygous B-NDG hFLT3LG, Flt3 KO mice.

Strain specific FLT3LG expression analysis in B-NDG hFLT3LG, Flt3 KO hFLT3LG mice by ELISA. Serum were isolated from B-NDG mice (n=3, 6-week-old) and homozygous B-NDG hFLT3LG, Flt3 KO mice (H/H) (n=3, 6-week-old). Expression level of mouse FLT3L and human FLT3LG were analyzed by ELISA (anti-mouse FLT3L ELISA kit: R&D, MFK00; anti-human FLT3LG ELISA kit: R&D, DFK00). Mouse FLT3L was detectable in B-NDG mice and homozygous B-NDG hFLT3LG, Flt3 KO mice. Human FLT3LG was exclusively detectable in homozygous B-NDG hFLT3LG, Flt3 KO mice. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in bone marrow by flow cytometry. Bone marrow cells were isolated from wild-type B-NDG mice (male, n=3, 6-week-old) and homozygous B-NDG hFLT3LG, Flt3 KO mice (male, n=3, 6-week-old). Flow cytometry analysis of the bone marrow cells was performed to assess the frequency of leukocyte subpopulations. Frequencies of T cells, B cells, NK cells, granulocytes, monocytes, macrophages in B-NDG hFLT3LG, Flt3 KO mice were similar to those in B-NDG mice. Frequencies of DCs in B-NDG hFLT3LG, Flt3 KO mice were lower than those in B-NDG mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.