Figure 1
Generation of a conditional human JAK2 V617F knock-in allele. (A) Diagram showing the endogenous mouse Jak2 allele, the targeting vector, the knock-in allele resulting from homologous recombination, and the activated recombined allele after excision of the PGKNeo-poly (A) cassette. (B) PCR showing high levels of recombination in BM, stem cells, and progenitors and relatively lower levels in peripheral blood, spleen, and thymus. PCR was performed using P1 + P2 + P4 (A) on genomic DNA from peripheral blood (PB), BM, LSK, total progenitors (Prog), LT-HSC (lineage−Sca-1+c-Kit+CD34−), ST-HSC (lineage−Sca-1+c-Kit+CD34+), myeloid progenitors (lineage−Sca-l−c-Kit+CD34+ CD71−), and erythroid progenitors (lineage−Sca-l−c-Kit+CD34− CD71+), thymus (Thy), and spleen cells (Sp). Serial dilutions were made by mixing the corresponding amount of genomic DNA from JAK2F/+ and JAK2R/+ ES cells. Top (P1 + P2) and bottom (P1 + P4) bands represent the recombined and floxed allele, respectively. (C) PCR (performed as in panel B) showing that the proportion of recombined allele in peripheral blood samples from JAK2V617F mice increases with time. Vertical lines have been inserted to indicate removal of gel lanes. (D) Real-time quantitative PCR analysis showing comparable up-regulation of Stat5 and Erk1/2 target genes in erythroid cells (both in fetal and adult erythroid cells) from JAK2V617F mice compared with results obtained from ET patients. In ET patient samples, individual BFU-E were genotyped, pooled according to genotypes, and transcript levels of target genes in JAK2 V617F mutant and wild-type colonies were compared (far right panel). The fold increase represents the ratio of gene expression in V617F and WT pools with each data point representing an individual patient. Transcript levels of the same target genes were increased by a similar amount in both fetal liver and adult BM BFU-E from JAK2V617F mice (V617F) compared with littermate controls (WT). BFU-E colonies were derived from 3 JAK2V617F and 3 wild-type control mice. Individual colonies from JAK2V617F mice were genotyped to distinguish those carrying the active recombined allele. Colonies were pooled according to the genotype (4-6 colonies/pool). Expression of target genes in a pooled BFU-E sample was calculated relative to the mean of the wild-type pooled samples, which was defined as 1. *P < .05. ***P < .001.

Generation of a conditional human JAK2 V617F knock-in allele. (A) Diagram showing the endogenous mouse Jak2 allele, the targeting vector, the knock-in allele resulting from homologous recombination, and the activated recombined allele after excision of the PGKNeo-poly (A) cassette. (B) PCR showing high levels of recombination in BM, stem cells, and progenitors and relatively lower levels in peripheral blood, spleen, and thymus. PCR was performed using P1 + P2 + P4 (A) on genomic DNA from peripheral blood (PB), BM, LSK, total progenitors (Prog), LT-HSC (lineage−Sca-1+c-Kit+CD34−), ST-HSC (lineage−Sca-1+c-Kit+CD34+), myeloid progenitors (lineage−Sca-l−c-Kit+CD34+ CD71−), and erythroid progenitors (lineage−Sca-l−c-Kit+CD34− CD71+), thymus (Thy), and spleen cells (Sp). Serial dilutions were made by mixing the corresponding amount of genomic DNA from JAK2F/+ and JAK2R/+ ES cells. Top (P1 + P2) and bottom (P1 + P4) bands represent the recombined and floxed allele, respectively. (C) PCR (performed as in panel B) showing that the proportion of recombined allele in peripheral blood samples from JAK2V617F mice increases with time. Vertical lines have been inserted to indicate removal of gel lanes. (D) Real-time quantitative PCR analysis showing comparable up-regulation of Stat5 and Erk1/2 target genes in erythroid cells (both in fetal and adult erythroid cells) from JAK2V617F mice compared with results obtained from ET patients. In ET patient samples, individual BFU-E were genotyped, pooled according to genotypes, and transcript levels of target genes in JAK2 V617F mutant and wild-type colonies were compared (far right panel). The fold increase represents the ratio of gene expression in V617F and WT pools with each data point representing an individual patient. Transcript levels of the same target genes were increased by a similar amount in both fetal liver and adult BM BFU-E from JAK2V617F mice (V617F) compared with littermate controls (WT). BFU-E colonies were derived from 3 JAK2V617F and 3 wild-type control mice. Individual colonies from JAK2V617F mice were genotyped to distinguish those carrying the active recombined allele. Colonies were pooled according to the genotype (4-6 colonies/pool). Expression of target genes in a pooled BFU-E sample was calculated relative to the mean of the wild-type pooled samples, which was defined as 1. *P < .05. ***P < .001.

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