Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

Abstract

Lentiviral transduction followed by puromycin selection is a well-recognized procedure for gene transfer and expression experiments using a variety of cell types including human hematopoietic stem and progenitor cells. Despite its widespread application, research regarding the potential effects of bacterial puromycin N-acetyltransferase (pac) gene expression in mammalian cell cultures is incomplete. Here the potential for puromycin selection to affect transcriptome profiles was examined using a well-studied model for human erythropoiesis. Experiments were performed using primary CD34(+) cells from six adult healthy human donors transduced with two commercially available pac-encoding lentiviral vectors and compared to non-transduced control cells. RNA-Seq gene expression profiles were generated at the proerythroblast stage of differentiation, then differential gene expression was analyzed with DEseq2 in R-Studio software. Inter-donor variation in the gene expression profiles and variations between puromycin selected populations after transduction of the separate lentiviral vectors was manifested by significant differences in the RNA detection levels of less than 0.1%. However, puromycin selection after pac gene transduction caused significant changes in over 5% of the mRNA when compared to non-transduced controls. The results suggest that consideration should be given for the potential of puromycin selection to confound the interpretation of RNA-Seq transcriptome profiles.

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“…This functional phenotype can be attributed to the cells overexpressing multidrug resistance gene(s) coding for ABC transporters able to expel puromycin from the cells (Theile, Staffen, & Weiss, 2010). Apart from the differences in the expression levels of endogenous gene(s) rendering puromycin resistance also unmatched intracellular levels of transgenic puromycin N -acetyltransferase and/or puromycin itself may distinctly affect cell transcriptomes (Guo, Lee, Byrnes, & Miller, 2017).…”