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Fig. 2 | Biomarker Research

Fig. 2

From: Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL

Fig. 2

Re-expression of KLF4 in PDX ALL cells using a tet-on inducible system. a, b KLF4 is downregulated in B-ALL cell lines, B-cell lymphoma cell lines and B-ALL PDX cells as compared to peripheral blood mononuclear cells (PBMC). a KLF4 mRNA was determined by RT-PCR using GAPDH as loading control; quantification of bands comparing KLF4 to GADH is depicted in the bar plot below. b KLF4 protein levels were analyzed by capillary immunoassay using β-Actin as loading control. KLF4 expression was normalized to the loading control and was plotted as the fold change relative to expression in PBMC. Mean ± SD of 3 independent experiments is shown. c KLF4 expression vector. The TRE promoter drives the expression of KLF4, which was either the wild-type (wt)KLF4 or a mutated (mut)KLF4 sequence lacking the two C-terminal zinc finger motifs comprising the DNA-binding domain (see Supplementary Figure S2B) that are each linked by a T2A peptide to the fluorochrome mCherry as a molecular marker; a mock vector (empty vector encoding only the mCherry fluorochrome) was used as a control. The addition of doxycycline (Dox; light green background throughout all figures) leads to the expression of the mock (gray), wtKLF4 (red) or mutKLF4 gene (blue; colors are identical throughout all figures) and mCherry (green). d Experimental design: Primary B-ALL cells from patients were transplanted into immunocompromised mice to generate the PDX models; PDX cells were consecutively transduced with three lentiviral constructs to express rtTA3 together with luciferase, tetR and either the mock, wtKLF4 or mutKLF4 gene (the vectors are detailed in panel C and Supplementary Figure S5A). Following passaging through mice for amplification, transgenic PDX cells were enriched by flow cytometry based on the constitutively expressed fluorochromes (mTaqBFP for the rtTA3-luciferase construct, iRFP720 or T-Sapphire for the tetR construct and Venus for the KLF4 constructs). Triple-transgenic cells were used for all further in vivo experiments. e PDX ALL-265 and PDX ALL-199 cells were infected as indicated in (d) and were cultured in vitro with or without the addition of Dox for 48 h, and KLF4 protein expression was analyzed by capillary immunoassays using PBMC from healthy donors as controls. β-Actin was used as a loading control. KLF4 expression is normalized to that of β-actin and is plotted as the fold change relative to the respective mock control. Mean ± SD of 3 independent experiments is shown

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