Characterization of the Jak2V617F KI mouse model. (A) Schematic representation of the targeting vector. Homologous recombination into the Jak2 WT allele of mouse embryonic stem (ES) cells resulted into the L2 knock-in (KI) genotype. A correctly targeted ES clone was injected into blastocyst stage embryos to generate L2 chimeric mice. Chimeras were bred with flippase (FLP) transgenic mice to generate L-. (B) PCR analysis of genomic tail biopsy DNA using primers a (5′-CCTGTCTCAGAATCCTTCTCATTTAGGG-3′) and b (5′-CTCCAGGGTTACACGAGTCACC-3′) detects successful recombination events in the L- KI mice (right panel). (C) Wild-type (WT) and mutated Jak2 Taqman allele specific amplification from bone marrow (BM) and spleen RNA samples of 2 KI (within the circle in plain line, studied in duplicate 12 and 20 weeks after birth) and WT mice (in duplicate, within the circle in dashed line) was performed. Standard curve (0%, 50%, and 100%) was carried out from mixtures of plasmids containing Jak2^V617F or Jak2^WT cDNAs. (D) Constitutive phosphorylation of ERK1/2 (Thr 202/Tyr 204) and STAT5 (Tyr 694) and total JAK2 protein in KI mice identified by Western blot analysis (antibodies from Ozyme). Cells isolated from WT and KI mice were starved for 40 hours and stimulated (+) or not (−) with interleukin-3 and granulocyte-macrophage colony-stimulating factor for 15 minutes. β-actin served as loading control. (Sigma). (E) Autonomous growth in KI mice represented as the percentage of CFU-Es derived from BM or spleen KI mice forming endogenous erythroid colonies (EECs) in the absence of added erythropoietin (mean value ± SD, n = 4). No EEC was detected from control mice. (F) Blood cell parameters (mean value ± SE) from KI (n = 11) and WT (n = 8) mice studied at 12 (± 2) weeks of age. RBC indicates red blood cell; MGV, mean globular volume; WBC, white blood cell; and MPV, mean platelet volume.