Priority Research Papers:

Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

Kevin Watanabe-Smith _, Jamila Godil, Anupriya Agarwal, Cristina Tognon and Brian Druker

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Oncotarget. 2017; 8:12596-12606. https://doi.org/10.18632/oncotarget.15392

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Kevin Watanabe-Smith1, Jamila Godil2, Anupriya Agarwal3,4, Cristina Tognon5,6 and Brian Druker3,6

1 Cancer Biology Program, Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA

2 Honors College, College of Science, Oregon State University, Corvallis, OR, USA

3 Division of Hematology and Medical Oncology, Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA

4 Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA

5 Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA

6 Howard Hughes Medical Institute, Portland, OR, USA

Correspondence to:

Kevin Watanabe-Smith, email:

Keywords: reproducibility in research, Ba/F3 transformation assay, functional validation, leukemia, oncogenes

Received: November 23, 2016 Accepted: February 12, 2017 Published: February 16, 2017


The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.

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