Generation of inducible Jak2V617F knock-in mice. (A) The targeted allele contains the floxed PGK-Neo-Stop cassette and the V617F mutation. This allele is transcriptionally silent, but can be induced in the presence of Cre to generate the activated Jak2V617F allele. (B) Constitutive phosphorylation of Stat5 in the BM of induced MxCre;V617F/+ and MxCre;V617F/V617F mice confirm expression of the mutant Jak2V617F protein. (C) Expression of total Jak2 mRNA was measured in the BM of WT, V617F/+, MxCre;V617F/+ (heterozygous), and MxCre;V617F/+V617F (homozygous) mice (n = 4) by real-time PCR. Total Jak2 mRNA expression was significantly reduced in the BM of both heterozygous and homozygous Jak2V617F mice compared with WT mice (P < .05 between WT and heterozygous Jak2V617F, P < .05 between WT and homozygous Jak2V617F; unpaired t test), whereas there were no significant differences between V617F/+ and heterozygous or V617F/+ and homozygous Jak2V617F mice. (D) A standard curve made from known ratios of accurately measured pMSCV-IRES-GFP plasmids containing mouse Jak2WT and mouse Jak2V617F showing the linearity and accuracy of the measurement of T/G fluorescence ratio (T-peak identifies the mutant, G-peak identifies the WT allele) for determination of allelic ratio. (E) Allelic ratio of the mutant Jak2V617F to WT Jak2 mRNA was determined by the T/G ratio after direct sequencing of the real-time PCR products from the BM of heterozygous Jak2V617F mice (n = 8). (F) The chromatograms of 3 sequenced real-time PCR products from the BM of heterozygous Jak2V617F mice are shown.