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. 2012 Jan 24;20(5):1002–1013. doi: 10.1038/mt.2011.298

Figure 1.

Figure 1

Characterization of lentiviral vector (LV)-driven dCK variant expression and prodrug activation in vitro. (a) Diagrammatic representation of LV expression constructs used in these studies. The transcription of the expression cassettes is driven off the elongation factor 1-α (EF1-α) promoter. The deoxyCytidine Kinase (dCK)-encoding vectors carry a bicistronic cassette, with the translation of the second cDNA, cytoplasmic tail-truncated human CD19 (huCD19Δ) marker, driven by the internal ribosomal entry site (IRES) derived from the encephalomyocarditis virus (EMCV). ψ, human immunodeficiency virus packaging signal; cPPT, central polypurine tract; EGFP, enhanced green fluorescent protein; LNGFRΔ, cytoplasmic tail-truncated low-affinity nerve growth factor receptor; LTR, long-terminal repeat; RRE, rev response element; SA, splice acceptor; SD, splice donor; SIN, self-inactivating LTR; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (b) Detection of overexpressed dCK enzyme by Western blotting of lysates from transduced Jurkat cells. Next, (c) Jurkat, (d) Molt-4, and (e) U87mg cells transduced with different LV/dCK constructs and sorted by fluorescence-activated cell sorting (FACS) were analyzed by flow cytometry for human CD19 expression (dotted line, nontransduced control cells; alternating dash/dot line, dCK.WT; dashed line, dCK.DM; solid line, dCK.DM.S74E). (f) Average provirus copy numbers in sorted populations of transduced Jurkat cells were quantified by WPRE-based quantitative-PCR (Q-PCR) and standardized against a cell line known to express a single proviral copy (error bars represent SE of the mean; n = 3; *indicates statistically significant difference, P < 0.005). (g) Lysates of transduced and nontransduced control Jurkat cells treated with 10 µmol/l bromovinyl-deoxyuridine (BVdU) for 12 hours were analyzed by high-performance liquid chromatography (HPLC) for intracellular accumulation of phosphorylated BVdU metabolites, immediate dCK product BVdU-monophosphate (BVdU-MP), BVdU-diphosphate and- triphosphate (BVdU-DP/TP) (error bars represent SD; n = 3; *indicates statistically significant difference, P < 0.005).