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. Author manuscript; available in PMC: 2019 Jan 1.
Published in final edited form as: Methods Mol Biol. 2019;1907:145–157. doi: 10.1007/978-1-4939-8967-6_12

Ex vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery

Kathryn A O’Donnell 1,2,*, Yabin Guo 3, Shruthy Suresh 1, Barrett L Updegraff 1, Xiaorong Zhou 1,4
PMCID: PMC6296821  NIHMSID: NIHMS933140  PMID: 30542998

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: Transposon mutagenesis, Sleeping Beauty, ex vivo forward genetic screening, cell-based screens, functional annotation, DNA transposons, growth-factor independence

1 Introduction

Large-scale genome sequencing efforts have identified numerous genetic lesions in human tumors. However, functional validation is still required for a significant fraction of recurrently mutated genes. Over the last decade, transposon-mediated mutagenesis has emerged as a powerful method for functionally annotating cancer genomes. A large number of studies have utilized transposon-based insertional mutagenesis screens in mice to identify and validate a number of genes relevant to tumorigenesis, including T-cell leukemia, colon cancer, hepatocellular carcinoma, medulloblastoma, and nerve sheath tumors [1–8]. To date, the vast majority of studies have relied upon the mobilization of mutagenic DNA transposons such as Sleeping Beauty (SB) or piggyBac in transgenic mouse models. When mobilized in the genome, these elements disrupt normal gene function by activating proto-oncogenes via a strong viral promoter, or by inactivating tumor suppressor genes via a transcriptional stop cassette. In these studies, the mutagenic transposon is mobilized at sufficient frequencies to induce tumorigenesis alone or in combination with an initiating event such as oncogenic activation of Myc, Braf, Npm, or genetic loss of Tp53, or Ptch tumor suppressors [7,9,10].

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. Transposon insertions sites are then recovered by high-throughput sequencing. We recently applied this approach to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells [11]. In this chapter, we provide a detailed protocol for performing ex vivo mutagenesis screens in these cells. We also provide a brief overview of genomic DNA isolation and sequencing of transposon insertions, although others have described similar methods for identifying transposon-tagged sequences using polymerase chain reaction-based strategies [12].

Unlike cDNA and RNA interference based screens, transposon-mediated mutagenesis generates both gain- and loss-of-function mutations in the same pool and/or tumor, which may more accurately model the process of tumor initiation, progression, or metastasis. Moreover, utilization of SB in ex vivo screening avoids local hopping, a phenomenon inherent to in vivo studies whereby the SB transposon favors transposition to sites adjacent to the donor concatemer in transgenic mice [13]. Finally, this ex vivo mutagenesis method may be adapted for performing screens in many different cell types and for distinct phenotypes with Sleeping Beauty or other transposon systems. Recently, somatic mutagenesis of human bone explant mesenchymal cells was performed with a hybrid lentiviral and SB mutagenesis system, generating myxofibrosarcomas in mice [14]. In contrast to this system where lentiviral vectors were used to introduce a SB transposon into the genome, our ex vivo approach relied solely on transient SB mobilization. Importantly, this system may be modified for conditional and dose-dependent expression of the transposon or transposase in different settings.

1.1 Cytokine signaling regulates growth and development of hematopoietic cells

Cytokine signaling is known to regulate the growth and development of hematopoietic cell populations [15–17] and it is well established that aberrant signaling through cytokine receptors and downstream pathways is an underlying cause of hematopoietic malignancies [18,19]. Thus, there is a strong precedence for using growth factor independence as a surrogate for transforming potential in leukemia. For example, genes that confer cytokine independence in murine bone-marrow derived Ba/F3 cells, which are normally dependent on IL3 for proliferation and viability, often exhibit broad transforming activity [20,21]. Constitutive activation of established oncogenes FLT3, ALK, or JAK3, or expression of TEL-JAK or EBF-PDGFRB gene fusions confers IL3 independent growth [22–25]. Ba/F3 cells have also been proven to be useful for validating oncogenes such as BCR-ABL and for characterizing the transforming potential of mutated kinases [26,27]. These prior studies demonstrated how the functional analysis of genes in these cells informs our understanding of the mechanisms of leukemogenesis. We performed a comprehensive analysis of genes that drive growth-factor independence and malignant transformation of Ba/F3 cells [11], which we hypothesized would reveal novel genes that promote signaling through the IL3 pathway (Figure 1) and that participate more broadly in hematopoietic malignancies and other cancers.

Figure 1. Overview of the IL3 signaling pathway.

Figure 1

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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.

2 Materials

2.1 Cell Culture

  1. RPMI1640 supplemented with 10% FBS (Invitrogen), 1% penicillin/streptomycin, and 10 ng/ml mouse IL3 (Cell Signaling Technology).

  2. Lonza Buffer SF plus supplement.

  3. Sleeping Beauty plasmids: SB11 or SB100X, and T2/Onc plasmids.

  4. Specialist equipment: 4-D Lonza Nucleofector, Tissue culture incubator, Biosafety cabinet, centrifuge, disposable pipettes, sterile filter tips, and pipettes.

2.2 Genomic DNA isolation

  • 4 × 96 well PureLink Pro 96 well Genomic DNA Purification Kit (includes all buffers)

  • Centrifuge that is capable of spinning 96-well plates at ≥ 2,100 × g, and accommodate a 7.0 cm microtiter plate stack, sterile filter tips, and pipettes.

3 Methods

3.1 Ex vivo Sleeping Beauty mutagenesis

  1. Warm RPMI1640 media supplemented with 10% FBS, 1% ml penicillin/streptomycin, and 10 ng/ml mouse IL3.

  2. Ba/F3 cells are cultured in the presence of IL3. To perform SB mutagenesis, spin Ba/F3 cells in a centrifuge at 100 × g for 5 minutes. 3–5 million cells are transfected in each Lonza cuvette (see Notes 1 and 2).

  3. Gently re-suspend cells with Lonza transfection solution (Buffer SF plus supplement). 100 μl buffer for each transfection.

  4. Carefully add 1 μg SB11 (or SB100X) plasmid and 1 μg T2/Onc plasmid to cells (see Note 3).

  5. For negative controls, transfect cells with 1 μg T2/Onc or SB plasmid alone (in separate cuvettes).

  6. Carefully pipette the mixture containing cells and plasmids to sterile cuvettes (see Note 4).

  7. Electroporate cells using Lonza nucleofector in a tissue culture biosafety cabinet using program DS-137.

  8. Allow cells to sit at room temperature for 10 minutes (in the tissue culture hood) immediately following electroporation.

  9. Add 1 ml pre-warmed media to cuvette and gently transfer cells to a flask (normally a T-25) with pre-warmed media (10 ml per T-25 flask). A typical concentration may be between 0.3 – 0.5 × 106 cells per ml of media.

  10. 72 hours after transfection, withdraw IL3 from media by replacing with fresh media (RPMI 1640, plus 10% FBS, and 1% penicillin/streptomycin (see Note 5). For a large-scale mutagenesis screen, cells from 1 T-25 flask may be split into a 48-well plate. The total volume of media is 200 μl/well in a 48-well plate. Culture cells in media lacking IL3 for 1–4 weeks (see Notes 6–7).

  11. Proceed to genomic DNA isolation from cells that grow in the absence of IL3. Alternatively, mutagenized IL3 independent Ba/F3 pools may be transplanted subcutaneously or by intravenous tail injection into immunocompromised NOD.Cg-PrkdcscidIl2rg−/−tm1Wjl/SzJ (NSG) mice (purchased from The Jackson Laboratories). Both methods of delivery robustly yield subcutaneous tumors or leukemias/lymphomas, respectively, within 2–8 weeks (see Note 8). An overview of a typical ex vivo mutagenesis screen is shown (Figure 2).

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

Figure 2

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(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.

3.2 Genomic DNA isolation

  1. For purification of genomic DNA from a high-throughput ex vivo mutagenesis screen, we follow the PureLink Pro 96 Genomic DNA standard protocol (Life Technologies). Set a water bath (or heat block) to 55°C (see Note 9).

  2. Add 20μl PureLink Pro 96 Proteinase K to each well of a 96 deep Well Block.

  3. Ba/F3 cells were harvested by centrifugation at 250 × g for 5 minutes. After removal of growth medium, resuspend cells in 200μl PBS.

  4. Transfer 200μl of sample (in PBS) to each well of a 96 Deep Well Block containing Proteinase K.

  5. Add 20μl RNase A (from kit) to each well. Seal the plate with Foil Tape. Mix well by briefly vortexing. Briefly centrifuge the plate to collect any lysate from the Foil Tape.

  6. Incubate at room temperature for 2 minutes.

  7. Remove Foil Tape and add 200μl PureLink Pro 96 Genomic Lysis/Binding Buffer. Seal the plate with Foil Tape. Mix well by vortexing to obtain a homogenous solution. Briefly centrifuge the plate to collect lysate.

  8. Incubate at 55°C for 10 minutes to digest protein.

  9. Remove Foil Tape and add 200μl 100% ethanol to the lysate. Seal the plate with Foil Tape. Mix well by vortexing to obtain a homogenous solution. Briefly centrifuge plate to collect lysate from the Foil Tape.

  10. Plate the PureLink gDNA Filter Plate onto a 96 Deep Well Block. Ensure that the nozzles of the gDNA Filter Plate extend into the wells of the 96 Deep Well Block.

  11. Transfer each lysate (~640μl) to a well of the gDNA Filter Plate using a multichannel pipettor. Unused wells should be covered with Foil Tape.

  12. Centrifuge the stacked plates at ≥2,100 × g for 5 minutes.

  13. Discard the flow through and reassemble the gDNA Filter Plate onto the used 96 Deep Well Block.

  14. Add 500μl Wash Buffer 1 (containing ethanol) into each well of the gDNA Filter Plate.

  15. Centrifuge the stacked plates at ≥2,100 × g for 2 minutes. Do not seal plate (this will ensure complete drying of the membrane).

  16. Discard the flow through and reassemble the gDNA Filter Plate onto a new 96 Deep Well Block (supplied in the kit).

  17. Add 50μl PureLink Pro 96 Genomic Elution Buffer to the center of the membrane in each well and incubate the plate for 1 minute at room temperature.

  18. Centrifuge the stacked plates at ≥2,100 × g for 3 minutes (see Note 10).

  19. Proceed to ligation mediated PCR, or cover wells with Foil Tape, and store purified gDNA at 4°C (short-term) or −20°C (long-term).

3.3 Identification of transposon insertion sites

  1. The junctions of the SB ends and Ba/F3 cell genomic DNA are amplified using ligation-mediated PCR (LM-PCR) [28]. Samples of genomic DNA are digested with BfaI and ligated to linkers (Figure 3A) (see Note 11).

  2. The ligation products are digested with BamHI and amplified by PCR with primers containing sequences of the T2/Onc left end and ligated linker; then a nested PCR is performed with sample-specific 8bp-barcoded primers (see Note 12).

  3. The PCR products are purified (using the MinElute 96 UP PCR Purification Kit, Qiagen) and eluted in a volume of 50 μl water. We typically run 2–5 μl on a 1% agarose gel to visualize transposon insertions (Figure 3B). Purified LM-PCR products are then quantified using a nanodrop and then pooled together into a single eppendorf tube (Figure 3C) (see Note 13).

  4. The PCR products are then sequenced using the Illumina HighSeq2000.

  5. The raw sequences are screened for barcodes and the T2/Onc end; then the T2/Onc sequence and linker sequence are trimmed and the genomic sequences aligned to the mouse genome using Bowtie (see Note 14).

  6. The alignment results are filtered to obtain the unique matches. The insertions are further filtered according to their frequency of occurrence. Any insertions with frequencies below 0.01% in their libraries are removed.

  7. The insertions from different libraries are pooled together and the insertions at same chromosome, coordinate, and orientation are collapsed to obtain non-redundant insertions.

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

Figure 3

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(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.

3.4 Identification of Common Insertion Sites (CISs)

We determined common insertion sites (CISs) by two different analyses (see Note 15).

Monte Carlo simulation

  1. The T2/Onc insertions are simulated by random distribution at TA sites throughout the mouse genome and repeated 1000 times. These represent possible sites for T2/Onc integration.

  2. Then the following “insertions in window size (bp)” were considered as significant (P<0.01) for our study [11]: 2 in 18 bp, 3 in 297 bp, 4 in 958 bp, 5 in 1895 bp, 6 in 3029 bp, 7 in 4309 bp, 8 in 5698 bp, 9 in 7175 bp, 10 in 8723 bp, 11 in 10330 bp.

  3. The CISs that overlap with each other are merged and the P values determined by comparing the insertion density in the CISs and the average insertion density in the entire genome using Pearson’s Chi-squared test.

Gene-centric CIS analysis

  1. For each of the genes in the mouse genome, the entire transcribed units and the region 10 kb upstream of the transcription sites are considered.

  2. The insertion densities were calculated by the following formula: Density= insertions in the gene/TAs in the gene/2.

  3. Then the densities are compared to the average density of the entire genome to determine the P values using Pearson’s Chi-squared test.

Acknowledgments

This work was supported by grants R01CA207763 from the National Cancer Institute (to K.A.O.), I-1881 from The Welch Foundation (to K.A.O.), SKF-15-067 from the Sidney Kimmel Foundation (to K.A.O.), 2015-03 from The LUNGevity Foundation (to K.A.O.), and RP150676 from the Cancer Prevention Research Institute of Texas (CPRIT, to K.A.O). We thank Jose Cabrero for assistance with the figures.

Footnotes

1

For Ba/F3 cell transfections, we utilized the 4-D Nucleofector System (Lonza) with Solution SF and Program DS-137. We found that the transfection efficiency of the pMAX-GFP plasmid in Ba/F3 cells was ~98% under these conditions. It is important to note that transfection conditions need to be optimized for each cell line, and methods other than electroporation are suitable.

2

Make sure to keep cells under sterile conditions at all times and use aerosol-resistant filter tips. Frequent spraying of hands and tissue culture hood with 70% ethanol before and after use is the best practice to avoid contamination of cells.

3

The T2/Onc element harbors splice acceptors and polyadenylation sequences in both orientations. Insertion of T2/Onc into a putative tumor suppressor will disrupt splicing and generate a loss-of-function mutation. The element also contains a murine stem cell virus (MSCV) LTR that contains promoter/enhancer elements followed by a splice donor that will facilitate splicing of transcripts initiated from MSCV to downstream exons. This allows for gain-of-function mutations as well as expression of truncated products. Although we used the T2/Onc element in our study, other there are additional SB transposons that may be utilized, including T2/Onc2 and T2/Onc3. A comparison between the different elements is reviewed in [29].

4

Pipette cells gently during the procedure as cells are fragile following electroporation. Avoid vigorous pipetting. Do not allow bubbles to form while cells and plasmid mixture are transferred to sterile electroporation cuvettes.

5

If desired, gDNA may be isolated from cells 48–72 hours after transfection (but prior to IL3 removal), and T2/Onc insertions sequenced to identify potential hot spots. In our experience, this was not a problem for our ex vivo screen, however identifying insertions from this pre-selection pool may be informative for downstream analyses.

6

It is important to freeze down aliquots of mutagenized (in this case, growth-factor independent) cells so that RNA and protein may be assessed, and downstream experiments performed after transposon insertions are identified.

7

Co-transfection with the T2/Onc and SB100X transposase plasmids, but not the individual constructs, resulted in the outgrowth of IL3 independent pools in approximately 20% of transfections after one to several weeks. The use of the SB11 transposase reduced the overall efficiency of growth-factor independence and degree of insertional mutagenesis. Fewer insertions may be desired depending on the specific genetic screen.

8

All procedures involving mice are approved by the Institutional Animal Care and Use Committeee (IACUC) of UT Southwestern Medical Center.

9

If fewer samples are analyzed and a 96-well format is not necessary, we often use another protocol for isolating high quality genomic DNA from cells and tumors that produces robust yields [12].

10

In our experience, a second elution with the PureLink Pro 96 Genomic Elution Buffer is not necessary. This will increase recovery but lower the overall gDNA concentration.

11

We typically perform ligation mediated-PCR assays using the left end of T2/Onc only in large-scale screens. If desired, one may perform LM-PCR assays and identify insertions from both ends of T2/Onc using BfaI for the left-end digestion and NlaIII for the right-end digestion, followed by linker ligation.

12

Nested PCR is performed using primers modified to include the sequence tags required for direct sequencing with the Illumina platform. We advise working with your institutional sequencing core to ensure that the most up-to-date sequencing tags are used.

13

In a typical Illumina run, we have pooled LM-PCR products from 3 × 96-well plates = 288 samples. Each sample is associated with a unique barcode. We pipette at least 25 ng of each LM-PCR product into a single tube, and the final concentration is adjusted to 20–25 ng/μl.

14

We utilized mouse genome mm10 for our mutagenesis screen in Ba/F3 cells, however we recommend using the latest build of human or mouse genome for mapping transposon insertions.

15

Although we utilized Monte Carlo Simulation analysis and Gene-Centric CIS analysis, there are other statistical approaches that may be applied, including Gaussian Kernel Convolution (GKC) and refined versions of the Poisson approach. A discussion of several different statistical methods is reviewed in [29]. It is recommended that investigators use multiple algorithms to identify CISs.

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