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. 2017 Oct 16;8(57):97231–97245. doi: 10.18632/oncotarget.21881

Figure 1. Production of a fibroblast library with shotgun gene mutations (retro-MEFs).

Figure 1

(A) Schema of the retroviral insertion sequences from the plasmids pMIG-LT and pLPC-TERT. (B) 293T cells (left panel) and freshly prepared MEFs (right panel) were transfected with pMIG-LT and cultured on slide for 24 h. Green fluorescence was assessed by fluorescence microscopy. Scale bars, 50 μm; data are representative of two independent experiments. (C) Agarose-gel images showing successful transfection. pMIG-LT was used to transfect 293T cells (lanes 1,3) or MEFs (lane 2,4); pLPC-TERT for MEFs (lanes 5,6). Inserted retroviral sequences were assessed by RT-PCR specific for GFP (amplicon size: 278 bp; lanes 1-2), retroviral LTR (amplicon size: 512 bp; lanes 3,4,5) or the puromycin-resistance gene (amplicon size: 441 bp; lane 6). M, marker. (D) Retro-MEFs were prepared by infecting MEFs with pMIG-LT (LT-GFP) or pLPC-TERT (TERT-puro) retrovirus. FB61 tumor cells were injected alone or in combination with retro-MEFs as indicated. Injection of retro-MEFs with pMIG-LT retroviral particles served as control. Tumor volumes were monitored over time as indicated. Mean ± SEM, **P< 0.01, two-way ANOVA, n=4 per group; data are representative of two independent experiments.