Figure 2. Outline of the ex vivo mutagenesis screen in Ba/F3 cells.

(A) In a typical ex vivo SB mutagenesis screen, cells are transfected with the mutagenic transposon (T2/Onc) and the SB transposase plasmids. As negative controls, cells are transfected with the individual transposon or transposase vectors. 48–72 hours post-transfection, IL3 is withdrawn from cells. This allowed for selection of Ba/F3 cells harboring transposon insertions in protein-coding genes or non-coding regions that confer growth-factor independence. After 2–3 weeks in culture, pools of IL3 independent cells were frozen down or transplanted into NSG mice and assayed for the development of leukemias and lymphomas in vivo. One advantage of the ex vivo approach is that transposon insertions may be sequenced at different times (prior to IL3 withdrawal, after pools became IL3 independent, and after tumors formed in mice). In a pilot screen, insertions in IL3 independent pools were compared to insertions present in tumors that developed in NSG mice. In the majority of cases, insertions that were present in the IL3 independent pools produced leukemias/lymphomas or subcutaneous tumors when injected into mice. Thus, IL3 independent growth in culture provided a useful surrogate for tumor-forming potential that allowed analysis of significantly larger numbers of independently mutagenized pools than could feasibly be analyzed following in vivo tumor growth. (B) Based on the results from the initial set of IL3 independent pools and tumors, we generated an additional 1,100 growth-factor independent pools through SB mutagenesis to comprehensively identify genes that contribute to cytokine signaling and B-cell transformation in this system. After generating a list of CIS genes, candidate genes were validated in vivo.