plasmid backbone pwpi  (Addgene inc)

Journal: Scientific Reports

Article Title: Gene-edited murine cell lines for propagation of chronic wasting disease prions

doi: 10.1038/s41598-019-47629-z

Figure Lengend Snippet: Stable reconstitution of PrP −/− cells with bank vole PrP (BV-PrP) using lentiviral transduction. ( a , d ) Immunofluorescence imaging of CAD5 and MEF reconstituted cells. Recombinant lentiviruses expressing bank vole PrP were generated using pWP1-BV plasmid which simultaneously expresses BV-PrP and EGFP. PrP −/− cells were transduced with these lentiviruses (CAD5_LV_BV and MEF_LV_BV) and the GFP + signature was visualized with Olympus IX51 fluorescence microscope. ( b , e ) Enrichment of CAD5 and MEF transduced cells by sorting top 30% GFP + hyper-expressors using FACS Aria cell sorter. The sorted cells were passaged and expanded into BV-PrP expressing stable cells. ( c , f ) Western blot showing stable integration and expression of bankvole PrP in CAD5 and MEF cells. WT represents the parental cells expressing mouse PrP and KO represents the PrP −/− cells. BV represents expression of bank vole PrP in the sorted and expanded cell population. The blots were probed with anti-PrP mAb 4H11 and actin was used as loading control.

Article Snippet: Since we were unable to get transductants using the pCDH construct, we changed the lentiviral backbone to pWP1 (Addgene#12254) which is a bicistronic vector that allowed simultaneous expression of BV-PrP and EGFP (pWP1-BV) under the EF-1α promoter (Fig. ).

Techniques: Transduction, Immunofluorescence, Imaging, Recombinant, Expressing, Generated, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Control