Transgenic expression of JAK2^V617F causes myeloproliferative disorders in mice

Construction of the transgene and generation of transgenic mice

The entire coding region of human JAK2^V617F plus the 3′ noncoding region were cloned into the HS321/45-vav vector provided by Dr Jerry M. Adams (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia).²³ The plasmid DNA was digested with SacII to remove the pBSIISK backbone and was used for injection into pronuclei of eggs from C57BL/6XDBA/2 F2 mice using the core facility at the Oklahoma Medical Research Foundation. The transgenic mice were identified by PCR amplification of tail genomic DNAs with primers TACAACCTCAGTGGGACAAAGAAGAAC and CCATGCCAACTGTTTAGCAACTTCA. The primers cover a 594 bp region in the coding sequence of JAK2^V617F. Transgenic founder mice were crossed with wild-type C57BL/6 mice. JAK2^V617F negative siblings of transgenic mice were used as controls throughout the study.

Analyses of mice

Peripheral blood samples (∼50 μL) were collected by tail bleeding into tubes containing potassium salt of ethylenediamine tetraacetic acid. Complete blood counts were obtained using a HESKA Vet ABC-Diff Hematology analyzer. Each analysis was done in duplicate. Blood samples with a platelet level of more than 2000 × 10⁹/L were diluted 3-fold in a RPMI medium containing 10% fetal bovine serum before analyses. Blood smears were stained with the May-Grünwald-Giemsa method. For histopathology analyses, femurs were fixed in formaldehyde, decalcified, and paraffin embedded. Spleens were treated in a similar way except that decalcification was omitted. Sections (4.5 μm) were cut and stained with hematoxylin and eosin. Reticulin staining was performed using the Accustain Reticulin Stain kit from Sigma-Aldrich (St Louis, MO). Levels of erythropoietin (Epo) and thrombopoitin (Tpo) in the plasma of mouse blood were determined using the mouse Epo and Tpo Quantikine enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN) according to the manufacturer's protocol.

Flow cytometric analysis and progenitor cell culture

Total white blood cells were obtained after lysis of peripheral blood with a red cell lysis buffer. Single-cell suspensions were prepared from bone marrow (from femurs) and spleen, and red cells were removed with the red cell lysis buffer. For flow cytometric analysis, the cells were stained with phycoerythrin-conjugated anti-B220 and fluorescein isothiocyanate-conjugated antibodies against Gr-1, Ter-119, CD71, and CD41 (BD Biosciences, San Jose, CA). The analyses were performed with a FACSCalibur Flow Cytometer at the Flow and Image Cytometry Laboratory of University of Oklahoma Health Sciences Center. All the cell cultures were carried out using media, growth factors, and cytokines from StemCell Technologies (Vancouver, BC). These included methylcellulose-based MethoCult and collagen-based MegaCult-C media. Cell cultures were done in triplicates, and colonies were scored after 2, 7, and 10 days for erythrocyte colony-forming unit, colony-forming units-megakaryocyte, and all other colonies, respectively, according to the manufacturer's protocols. Bone marrow stromal cells were cultured and expanded in the MesenCult Medium (Mouse) with Mesenchymal Stem Cell Stimulatory Supplements. The second passage cells were harvested for isolation of total RNAs.

Real-time polymerase chain reaction analysis

Total RNAs were isolated from peripheral blood, bone marrow, spleen, stromal cells, and various other tissues of mice using the Trizol reagent (Invitrogen, Carlsbad, CA) and were then treated with RQ1 RNase-free DNase (Promega, Madison, WI) to remove contaminated genomic DNAs. First-strand cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Real-time polymerase chain reaction (PCR) was performed in an IQ5 Multicolor Real-Time PCR Detection System using iQ SYBR Green Supermix (Bio-Rad). PCR amplifications were performed in triplicate with multiple dilutions for human JAK2^V617F and mouse Jak2 along with parallel measurements of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (an internal control). To confirm specific amplification of desired PCR product, melting curves were analyzed and PCR products were separated on a 3.0% agarose gel. As an additional control, reverse transcription (RT–PCR) was also performed with equivalent amounts of RNA samples without reverse transcription. The following primers were used for PCR amplification: Mouse GAPDH, ACTCCCACTCTTCCACCTTCG (forward) and CACCCTGTTGCTGTAGCCGTA (reverse); human JAK2^V617F, AGATGTGAAAATATCTGCTCAAAACT (forward) and AAACTTCTTACAAAATCCTTGCTAAG (reverse); mouse Jak2, AGACTTCCAGAACCAGAACAAAG (forward), and TCACAGTTTCTTCTGCCTAGCTA (reverse). The primer sets for human JAK2 and mouse Jak2 were derived from the last exons corresponding to the 3′noncoding regions of each cDNA where the 2 genes share essentially no sequence identity. The primers work for cDNAs as well as genomic DNAs. The PCR conditions were 95°C for 20 seconds, 59°C for 20 seconds, 72°C for 20 seconds, for 45 cycles. To determine the copy number of transgenic JAK2^V617F, PCR products of human JAK2 and mouse Jak2 amplified with the above primer sets were cloned into the pBluescript KS vector, and the purified plasmid DNAs, with concentration being accurately measured, were used as standards in real-time PCR analyses. The absolute amounts of human JAK2^V617F and mouse Jak2 in a given DNA samples were thereby determined, and the ratios of these gene DNAs represent relative gene copy numbers.

Statistical analysis

Statistical analyses were performed using the Excel program. Differences between 2 groups of samples were accessed using t tests. P values less than .05 (2-tailed) are considered significantly different.

Image acquisition

Slides were viewed with an Olympus BX-51 upright microscope equipped with U Plan Fluorite objectives at 40×/0.75 and 100× oil/1.30 (Olympus, Tokyo, Japan) and Cytoseal mounting medium (Richard-Allan Scientific, Kalamazoo, MI). Images were acquired using a DP71 digital camera with DP-BSW-V3.1 camera control software (Olympus) and were processed with Adobe Photoshop version 7.0 software (Adobe Systems, San Jose, CA).

Generation of JAK2^V617F transgenic mice

By transplanting bone marrow cells expressing JAK2^V617F mediated by retroviral vectors, several studies have shown that the JAK2 mutant produces PV-like phenotype in mice.^11–14 These studies have verified the pathogenicity of JAK2^V617F. However, these mouse models are not permanent mouse lines. To create a stable mouse MPD model, we have chosen a transgenic approach. Considering the general effects of JAK2 on the development of cells other than hematopoietic cells, we chose a HS321/45-vav vector, which uses the vav gene promoter to control gene expression. Based on the previous studies, the vav promoter drove expression of human CD4 efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues, including B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid, but not in nonhematopoietic cell types.²³ By following a similar procedure, we generated 3 JAK2^V617F transgenic founder mice, designated AF0, BF0, and CF0, which give rise to A, B, and C lines of progeny, respectively. The AF0 and BF0 mice are viable and fertile. AF0 became very inactive and essentially moribund at 69 weeks and was thus killed for analyses. BF0 died at 93 weeks. Both A and B founder mice displayed enlarged spleens. CF0 did not look healthy from the beginning. It died suddenly at the age of 4 weeks when it was picked up from the back. The cause of death remains unknown. It also had an enlarged spleen. The founder line transgenic mice have the C57BL/6XDBA hybrid background, and they were crossed with wild-type C57BL/6 mice to reach purer genetic background. This study was carried out with F0 to F6 generation mice.

Analyses of transgenic JAK2^V617F gene expression

We used real-time PCR analyses to determine the gene copy numbers of JAK2^V617F in mice. Our real-time PCR analyses yielded products showing single bands of expected size on agarose gels and single peaks on melting curve analyses (data not shown). Using purified plasmid DNAs as standards, we first generated linear standard curves as shown in Figure S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Based on the standard curves, we were able to determine the absolute amounts of human JAK2 and mouse Jak2 DNAs in a given sample and thereby calculate copy numbers of the JAK2^V617F transgene (Figure 1A). The gene copy numbers are 13 plus or minus 1.4, 1.9 plus or minus 0.3, and 24 plus or minus 2.7 for the A, B, and C founder mice, respectively. Over 6 generations, the A and B line mice kept the same transgene gene copy numbers as their founder mice. This implies that the JAK2^V617F transgene was inserted into a single chromosome in each mouse line, likely through one integration site. This was further supported by the fact that all JAK2^V617F mice were born with the expected Mendelian ratio. To verify the tissue specificity of the vav promoter we used, we analyzed the mRNA level of JAK2^V617F in various tissues of line A mice. As shown in Figure 1B, exogenously introduced human JAK2^V617F is expressed in hematopoietic tissues, including thymus, bone marrow, spleen, peripheral blood, and lymph nodes, but hardly at all in various nonhematopoietic tissues except for lung. Interestingly, endogenous mouse Jak2 is highly expressed in all the hematologic tissues but also at a significant level in other tissues, such as lung, ovary, intestine, uterus, and stomach (Figure S2). We further analyzed the expression of JAK2^V617F in bone marrow stromal cells obtained from in vitro culture. The stromal cells also expressed the JAK2^V617F transgene gene, but at a level approximately one-tenth of that seen in the whole bone marrow cells (Figure 1B). A contribution of this expression to the observed phenotypes described in “Phenotypes of JAK2^V617F mice” may be less likely but cannot be totally ruled out. We also compared the relative expression levels of JAK2^V617F mRNA in A and B line mice. On average, JAK2^V617F expression in line A mice was 4 times higher than that in line B, which is correlated with the gene copy numbers (data not shown). The fact that mRNA levels were not directly proportional to the gene copy numbers may be the result of the so-called repeat-induced gene silencing effect seen in transgenic animals with multiple transgene copies in concatameric arrays.²⁴ However, within each line of mice, the expression level is consistent among different members over generations, despite some variations in phenotypes. With the standard curves shown in Figure S1, we were also able to calculate the ratio of reversely transcribed cDNAs of JAK2^V617F and mouse Jak2 in a given sample. With bone marrow, spleen, and peripheral blood samples, the cDNA level of JAK2^V617F was no more than 1/16th that of mouse Jak2, even for the line A transgenic mice. Nonetheless, this result should not be overinterpreted. Because the reverse transcription efficiencies for mJak2 and hJAK2^V617F may be different, the data do not necessarily mean that the mRNA level of human JAK2^V617F is less than 1/16th of that of mouse JAK2. In addition, even if the hJAK2 mRNA is that much lower, it does not necessarily produce less JAK2^V617F protein because the translation efficiencies of these 2 mRNAs may also be different.

We attempted to use Western blotting analyses to detect the expression of JAK2^V617F in white blood cells from peripheral blood, bone marrow, and spleen. The antibody worked well, but samples from transgenic mice did not show a significantly higher level of JAK2 expression over the basal level of mouse JAK2 in control mice (data not shown). So far, we don't have a specific antibody that recognizes human JAK2 but not mouse JAK2. The data suggest that the overall expression level of exogenously introduced JAK2^V617F was not very high, further suggesting that the vav promoter is a relatively mild promoter as previously reported.²³ We then performed cell staining to detect expression of JAK2^V617F in individual cells. Figure 2A shows that a small fraction (<1%) of peripheral blood white cells expressed a high level of JAK2. In consistence with the mRNA expression levels, cells from line A mice appeared to be stained brighter than those of the line B mice. We observed a similar expression pattern of JAK2^V617F in cells from bone marrow and spleen (not shown). This explains why Western blotting analyses of whole cell extracts failed to show a significant increase in the JAK2 protein level in transgenic mice. The nature of these positively stained cells is to be determined. We further detected JAK2 kinase activity in the extracts of mouse tissues using a sensitive assay system recently developed.^25,26 As shown in Figure 2B, a markedly increased phosphorylation of GST-JAKS was seen in samples from transgenic mice compared with the control. This increased activity is presumably attributable to the expression of JAK2^V617F. As a deregulated enzyme, JAK2^V617F was known to cause constitutive cell signaling as revealed by many studies.^5–14 This was found to be the case as illustrated by a significantly increased level of ERK2 phosphorylation in bone marrow cells of line A mice in comparison with the control mice (Figure 2C). We also analyzed phospho-ERK2 in line B mice and phospho-Akt and phospho-STAT5 in all the transgenic mice but detected only subtle increases over the control mice (data not shown).

Phenotypes of JAK2^V617F mice

We have performed complete blood counts for more than 200 transgenic mice and their nontransgenic siblings and observed significant increases in blood counts, particularly for the line A mice. Table 1 summarizes data obtained with mice 10 to 12 weeks of age. Compared with the nontransgenic control mice, line A transgenic mice demonstrated markedly increased counts of white cells, red cells, hemoglobin, hematocrit, and platelets. To a lesser degree, the line B mice which had a lower level of JAK2^V617F expression also produced increased blood counts. Red blood cell counts, hemoglobin, hematocrit, and platelet counts in the line B mice are considerably higher than the control mice, although their white cell counts were not significantly different. Interestingly, statistical analyses revealed that mean corpuscular volume and mean corpuscular hemoglobin of red cells from transgenic mice, especially line A, were significantly below those of control mice. Staining of blood smears revealed that the shape of red cells is apparently normal, but line A mice have many obviously smaller red cells (Figure 3A). Line A transgenic mice also displayed a markedly increased number of platelets in the blood smear with the occasional presence of giant platelets. Within the line A mice, approximately 75% showed strong elevations in the platelets (usually 3000 × 10⁹/L) but with normal or slightly increased counts in white cells and red cells. The phenotype of these mice resembles that of human ET patients. The remaining approximately 25% of line A mice displayed abnormally high levels of red blood cells and white blood cells as well as platelet. This puts the mice into the PV category. Overall, essentially all the line A mice had elevated red cells, white cell, and/or platelets. Many line B transgenic mice exhibited moderately elevated blood counts, which did not reach levels that can be defined as ET or PV. The data provided in Table 1 were obtained from F2 to F6 generation mice at 10 to 12 weeks of age. Normal variation may contribute to the differences in phenotypes observed within each line of mice, although genetic background may be a factor as well. Age also affected blood counts of mice. Figure 3B illustrates several typical cases. Some mice started with high blood counts at the age of 4 weeks, whereas others started with normal or near-normal counts but increased to abnormal levels over time. Some mice showed a continual increase in blood counts over a long period, whereas others were stabilized or even showed some decline. So far, none of these mice have had their blood level decline to a point of anemia or thrombocytopenia. Finally, male and female mice appeared to respond similarly to transgenic expression of JAK2^V617F.

Flow cytometric analyses were conducted with single cell suspension of peripheral blood, bone marrow, and spleen cells from nontransgenic and line A transgenic mice. Forward and side scattering data revealed a profound increase in the proportion of nonlymphocytic cells in these 3 tissues (not shown). Staining of cells with anti-B220, a B-cell marker, and anti–Gr-1, a myeloid cell marker, verified the elevation in granulocytes in all 3 hematopoietic tissues (Figure 3C). Further staining of spleen cells with anti-Ter-119 and anti-CD71 for erythroid cells and anti-CD41 for megakaryocytic cells demonstrated erythroid and megakaryocytic hyperplasia in the spleen and bone marrow (Figure 3D).

Splenomegaly is also a clear phenotype of the JAK2^V617F transgenic mice (Figure 4). Overall, the line A mice have strikingly enlarged spleens, weighing 2 to 8 times more than those of control mice. Line B mice also exhibited enlarged spleens but to a much lesser degree, which is consistent with their relatively moderate elevations in blood cell counts. The line C founder mouse, which died at the age of 4 weeks, had a spleen 10 times the size of that of control mice of comparable ages.

Histologic staining of bone marrow and spleen is shown in Figure 5A. All the transgenic mice displayed marrow megakaryocytic hyperplasia, most remarkably for line A and C mice in which the marrow displayed a remarkably increased number of megakaryocytes. The spleens of the transgenic mice were also infiltrated with megakaryocytes, which were not found with the control mice. We also performed reticulin staining to detect development of myelofibrosis in the transgenic mice (Figure 5B). Among 26 line A mice analyzed, 10 developed various degrees of fibrosis in the bone marrow and spleen. These 10 mice were all over the age of 30 weeks and showed more severe PV-like or ET-like phenotypes than those without fibrosis. Interestingly, fibrosis was not found in 10 of the line A mice, which were younger than 30 weeks, suggesting a strong correlation between fibrosis and aging. Figure 5B illustrates results obtained with the 69-week-old line A founder mouse, which had elevated platelet counts only, and a 35-week-old F2 generation line A mouse, which displayed elevations in red cell, white cell, and platelet counts. The data demonstrated clear fibrosis in both bone marrow and spleen, which was not present in the control mice. So far, we have not discovered the occurrence of fibrosis in more than a dozen of line B mice except for the founder mouse, which died at the age of 93 weeks with an enlarged spleen (data not shown).

We further performed hematopoietic colony assays to compare the number and the property of progenitor cells in the peripheral blood, spleen, and bone marrow from control and line A transgenic mice. The data are shown in Figure 6. When cultured with optimal concentrations of growth factor and cytokines, peripheral blood from line A transgenic mice produced a significant number of colonies, which were essentially not found with the control mice. This indicates an occurrence of stem-progenitor mobilization in the transgenic mice, a feature also found in MPD patients. The bone marrow and spleen from the transgenic mice also gave rise to much increased colonies, indicating the presence of a larger percentage of progenitor cells in these tissues. For the bone marrow, however, the total number of progenitor cells in the transgenic mice may be reduced because the total nucleated cells obtained from their femurs were usually less than 50% of those from control wild-type mice. We have also cultured bone marrow cells in the absence of any added growth factors and cytokines, and the data shown in Figure 6A demonstrate that cells from line A transgenic mice produced a significant number of granulocyte-macrophage colony-forming cell colonies, whereas control cells hardly formed any colony. A hallmark of MPDs is autonomous growth of hematopoietic progenitor cells in vitro in the absence of growth factors. This was found to be true for the JAK2^V617F transgenic mice. Figure 6B demonstrates autonomous formation of erythrocyte colony-forming unit colonies with cells from bone marrow of line A transgenic mice. Analyses with the MegaCult-C collagen-based media also demonstrated a much increased number of megakaryocytic progenitor cells in the bone marrow of line A transgenic mice and the ability of the cells to form colonies in the absence of Tpo (Figure 6C). Figure 6 shows data obtained with line A mice only. With line B mice, the trend is the same, although the changes were less remarkable (data not shown).

Finally, a major feature of PV is a reduced level of Epo in the blood serum.^1–3 This was also found in our JAK2^V617F transgenic mice, especially with the line A mice (Figure 7A). Indeed, the average level of Epo in line A mice was less than one fourth that found in control mice. Epo levels found in individual line A mice ranged from 9 to 80 pg/mL compared with 115 to 180 pg/mL found with control mice. To a lesser extent, line B mice also showed a significant reduction in their plasma Epo. We further analyzed the plasma level of Tpo (Figure 7B). Whereas the line A mice displayed a profound decrease in the Tpo level, the line B mice did not show a significant change. In all, the reduced levels of Epo and Tpo are inversely correlated with the increased red blood cell and platelet counts in transgenic mice. This presumably reflects a feedback inhibition on the syntheses of Epo and Tpo by the increased red cells and platelets in the peripheral blood of transgenic mice.