lentiviral pwpi backbone  (Addgene inc)

Journal: Journal of Human Immunity

Article Title: A heterozygous USB1 variant linked to immunodeficiency

doi: 10.70962/jhi.20250110

Figure Lengend Snippet: The USB1 de novo variant is catalytically active and correctly processes U6 snRNA. (A) Total RNA extracted from the indicated BEBV cell lines were treated with T4 PNK or with buffer only (PNKBuff) in mild acidic conditions. RNA was subsequently treated with poly(A) polymerase (PAP). Nontreated RNA was loaded as a control, ( n = 2). (B) 3′ RACE analysis of U6 oligo(U) tails in the indicated cell lines. At least 24 clones per sample in each experiment ( n = 2) were sequenced. Bars and error bars are averages of the number of U's within U6 oligo(U) tails and SEM from two independent experiments. (C and D) Indicated cell lines were treated with actinomycin D for 0, 4, and 8 h. RNA samples were processed by northern blotting for detection of U6 and 5S ( n = 2). L: marker of known length (67 nucleotides). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. (E and F) U6 relative abundance quantification by qPCR analysis on patients and control cell lines (ctr1 n = 2, ctr2 n = 3, ctr3 n = 1, USB1 −/− n = 3, P1 n = 3) (E), and USB1 −/− cells transduced with the indicated lentiviral constructs ( n = 2) (F). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. EV, empty vector. Source data are available for this figure: .

Article Snippet: Those inserts (with the addition of an HA tag at the C-terminal when indicated) were subcloned into the lentiviral pWPI backbone (RRID:Addgene_12254) by GenScript.

Techniques: Variant Assay, Control, Clone Assay, Northern Blot, Marker, Transduction, Construct, Plasmid Preparation

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