Generation of a mouse model with FLT3^wt/ITDmutation. (A) Strategy for targeted insertion of an ITD mutation into murine Flt3 genomic DNA. The targeting vector was a 12-kb fragment flanking exons 10 to 16 of murine FLT3 genomic DNA. An 18-bp ITD mutation was inserted into exon 14. MC1-TK cassette and LoxP-flanked PGK-Neo cassette were inserted as indicated into the targeting vector, acting as negative and positive selection markers, respectively. The sites of diagnostic restriction enzyme KpnI (K) and ProbeA used for Southern Blotting analysis are shown; ▵ indicates LoxP. (B) Homologous recombination and presence of ITD mutation in FLT3^wt/ITD mice was confirmed by Southern blotting analysis (i) and PCR analysis (ii). (Bi) Southern blotting analysis confirmed the presence of homologous recombination on one allele of Flt3 genomic DNA. (Bii) PCR analysis detected the presence of homologously recombined allele (top panel) and ITD mutation (bottom panel). (C) PIPC-treated FLT3^wt/ITD mice showed enhanced FLT3 expression. (Ci) RT-PCR detected transcription of both ITD and wild-type FLT3 in BM cells from FLT3^wt/ITD mice; numbers shown below each lane in the top panel are the mean ratio of the ITD (top band) versus wild-type (bottom band) FLT3 expression determined by densitometry analysis. (Cii) Quantitative RT-PCR showed increased expression of FLT3 in FLT3^wt/ITD mice. The level of total FLT3 expression was normalized based on that of mS16. Data are expressed as means (bars) plus or minus SEM (error bars). (Di) Western blotting analysis showed enhanced FLT3 activity and expression in FLT3^wt/ITD mice. Lysed spleen cells were immunoprecipitated with EB-10 antibody and immunoblotted using 4G10 (top panel) or anti-murine FLT3 antibodies (bottom panel). (Dii) Flow cytometric analysis shows elevated FLT3 expression in the Lin^−/low population of the BM from FLT3^wt/ITD mice. wt indicates wild-type.