Abstract

It is generally assumed that the JAK2-V617F mutation is the cause of disease in a large proportion of patients with myeloproliferative disorders. However, recent evidence suggests that additional genetic alterations might be collaborating with JAK2-V617F. Most importantly, screenings of hematopoietic cells from JAK2-V617F positive patients using quantitative PCR-based methods have revealed a strong variability in the fraction of cells that carry the mutation and in some patients a striking discrepancy with the extent of clonal hematopoiesis. On the other hand, retroviral overexpression of JAK2-V617F in mouse bone marrow transplantation models causes phenotypes that resemble human polycythemia vera, the MPD entity in which the incidence of JAK2-V617F is highest. In the mouse transplantation models described to date, retroviral vectors were used that bear the risk of insertional mutagenesis and in most cases lead to a strong overexpression of JAK2-V617F. These limitations might hamper an accurate reproduction of the human disease. To express the mutant JAK2 at physiological levels in a mouse model, we generated JAK2-V617F transgenic mice. The transgene was constructed based on a human BAC containing JAK2 exons 1–12 and approximately 100 kb of upstream sequence. A cDNA fragment covering exons 13–25 of JAK2, which includes the V617F mutation, was appended by homologous recombination in bacteria. We also generated an identical construct with the JAK2 wild type sequence as a control. Pronuclear microinjection of the control construct yielded 8 viable and fertile founders with transgene copy numbers ranging from 1 to 18. We also obtained 3 mice that were positive for the JAK2-V617F transgene. One of these mice died 2 days after birth, a second mouse died at the age of 5 weeks. This mouse had 3 integrated copies of the transgene and interestingly an approximately 10-fold enlarged spleen. Unfortunately, we were unable to obtain additional data from this mouse. The third mouse with one integrated copy of the transgene was viable and gave rise to a transgenic line (VF1). The VF1 line expresses the transgene at approximately 25% of the endogenous wild-type Jak2 level. We analyzed blood counts at the age of 5–6 weeks and observed a small but significant elevation of platelet numbers (approximately 1.2-fold), which was reproduced when transplanting JAK2-VF1 bone marrow into wild type recipients. No changes in hematocrit or white blood cell count were detected. The low yield of JAK2-V617F transgenic mice together with the early death of two of the founders suggested that the transgene could be lethal when expression exceeds a certain level. To overcome this potential problem and to allow for inducible expression of the JAK2-V617F transgene, we generated a second construct in which exons 13–25 of the original transgene were inverted to abolish expression of catalytically active Jak2 in this configuration. Antiparallel loxP sites were placed at the borders of this inverted fragment to allow Cre-mediated repair of the transgene. These loxP sites contain previously characterized mutations to prevent continuous recombination. We have obtained 4 founders of this Cre-activated JAK2-V617F transgene with copy numbers between 1 and 9. After crossing to Mx-Cre mice and inducing Cre we observed repair of the transgene at the DNA level. Furthermore, mRNA of the recombined JAK2-V617F allele was detectable in peripheral blood. The effects of the JAK2-V617F transgene induction on the hematopoietic system are currently being studied in detail and will be presented.

Disclosure: No relevant conflicts of interest to declare.

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