Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery

Abstract

Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability. This approach allows discovery of both gain- and loss-of-function mutations in the same screen. Transposon insertions sites are recovered by high-throughput sequencing. We recently applied this system to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells. Here we describe a method for performing ex vivo Sleeping Beauty-mediated mutagenesis screens in these cells, which may be adapted for the acquisition of many different phenotypes in distinct cell types.

Keywords: Cell-based screens; DNA transposons; Ex vivo forward genetic screening; Functional annotation; Growth-factor independence; Sleeping Beauty; Transposon mutagenesis.

Figure 1. Overview of the IL3 signaling pathway

IL3 is a cytokine that regulates the growth and differentiation of hematopoietic cell populations. It is a growth factor for B lymphocytes, and promotes survival for a number of different cell types. When IL3 binds to its heterodimeric receptor, this leads to activation of downstream pathways including JAK/STAT, PI-3K/AKT and MAPK. We hypothesized that comprehensive mutagenesis of Ba/F3 cells, which normally require IL3 for growth, would reveal novel genes that promote signaling through the IL3 pathway.

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cells are transfected with the individual transposon or transposase vectors. 48–72 hours post-transfection, IL3 is withdrawn from cells.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.

Figure 3. Ligation-mediated PCR results from an ex vivo SB-mediated mutagenesis screen

(A) Genomic DNA from cells and tumors was digested and used to perform ligation-mediated PCR to amplify transposon-genomic junction fragments. (B) Ligation-mediated PCR products from a Ba/F3 pilot mutagenesis screen were run on an agarose gel. TV and SC refer to the route of cell administration for tumors that developed in mice. TV = tail vein (tumor tissue isolated from spleen); SC = subcutaneous tumor. Whereas some LM-PCR reactions produced a smear of PCR products, dominant bands were observed in the IL3 independent pools and in tumors. (C) After pooling of all LM-PCR products in one tube, a smear of products (representing many genomic transposon insertions) was observed.