JAK2 is not required for red cell formation by BCR-ABL.

(A) Retroviral infection of JAK2^−/− fetal liver cells: E12.5 JAK2^−/− fetal livers contain very few cells (< 2 × 10⁵ cells/embryo)5 and are unresponsive to many cytokines used in retroviral infection.6 The protocol detailed here provided optimum conditions for retroviral infection of JAK2^−/− fetal liver cells. (B) Flow cytometry analysis of transduced JAK2^−/− fetal liver cells, a representative analysis of 10 independent experiments performed. E12.5 JAK2^−/− fetal liver cells were infected with the control vector MIG-control or with the bicistronic retroviral vectors MIG-P210 or MIG-JAK2 in the presence of SF and IL-6 alone (MIG-control and MIG-P210) or together with Epo added 12 hours after initiation of infection (MIG-JAK2). Cells were analyzed 60 hours later for their expression of GFP and the erythroid marker Ter119-PE; 100 000 live events were collected. The percentage of Ter119⁺ (i) and Ter119⁻ (ii) cells within the population of GFP⁺ cells are indicated in each case. (C) GFP-positive cells (panel B, populations i and ii) of JAK2^−/− fetal liver cells transduced with MIG-P210 and cultured in the absence of Epo (Ci, ii) or with MIG-JAK2 and cultured in the presence of Epo (iii, iv) were isolated by FACS and analyzed for their morphology by Wright Giemsa staining. Representative fields are depicted (× 1000). Arrows (Cii, iii) show an enucleated erythrocyte derived from a P210-transduced cell (Cii), or an orthochromatic erythroblast derived from a JAK2-transduced cell cultured with Epo, in the process of exonucleation (Ciii). Control MIG-transduced cells did not generate enough cells to perform morphologic analyses routinely. (D) A representative field of Wright Giemsa analysis of freshly isolated JAK2^−/− fetal liver cells (× 1000). ND indicates not determined.