pcdna4to-scn8a-variant-3-ires-mscarlet  (Addgene inc)

Journal: The Journal of Cell Biology

Article Title: Translation of unspliced retroviral genomic RNA in the host cell is regulated in both space and time

doi: 10.1083/jcb.202405075

Figure Lengend Snippet: Visualization of viral FL RNA translation by dual RNA-Gag labeling. (A) Schematic representation of the MLV construct. MLV-ST-MS2 contains an in-frame insertion of the 24XST in Gag and the 24XMS2 in Pol. Ѱ refers to the RNA encapsidation signal, splice donor (SD, SD’) and acceptor (SA) sites are shown, as well as the start codons for Gag (ATG) and for gGag (CTG). (B) Representative images of a GFP/RFP cell expressing WT MLV-ST-MS2 acquired on a widefield microscope. Merge and maximum intensity projection (MIP) of several z-stacks are presented on the left. A cell treated with puromycin is shown in the lower panels. In the zoom insets, red arrows indicate non-translating RNA, yellow arrows indicate translating RNA, and green arrows indicate mature Gag. Scale bars are 10 and 1 µm for zoom insets. (C) Quantification of MS2 and ST spots per cell detected with Imaris in the presence or absence of puromycin. (D) Proportion of MS2 spots colocated with ST spots per cell. For all graphs, the mean and SEM are shown with n = 38 cells. The significance of differences was assessed using a nonparametric test (Mann–Whitney) (ns = nonsignificant, ****P ≤ 0.0001).

Article Snippet: The sequence of 24XST repeats of the plasmid pcDNA4TO-mito-mCherry-24XGCN4 (Addgene_60913) was amplified by PCR (forward: 5′-CCT CAC TCC TGG CGC GGC CGC GGG AGG TTC TGG AGG ATT GT-3′ and reverse: 5′-GGC GCC TAG AGA CGC GGC CGC GCC GCC AGA CCC TCC TCG-3′) and then inserted in place of EGFP into the Gag MLV subclone pcDNA.MLVgp.EGFP.p12 (a gift from C. Baum [ ]) opened with NotI by using the HiFi assembly kit (NEB).

Techniques: Labeling, Construct, Expressing, Microscopy, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Translation of unspliced retroviral genomic RNA in the host cell is regulated in both space and time

doi: 10.1083/jcb.202405075

Figure Lengend Snippet: Parameters used to determine the number of ribosomes translating MLV FL RNA. (A) Illustration of different intensities of fluorescence of a single ribosome depending on its position on FL RNA. Lengths of Gag-24ST domains are given in amino acids. Created with https://BioRender.com . (B) KIF1C calibrator fused to 24XST at C terminus. N is the length of the protein counting from ST region and n corresponds to the length of 24XST in amino acids.

Article Snippet: The sequence of 24XST repeats of the plasmid pcDNA4TO-mito-mCherry-24XGCN4 (Addgene_60913) was amplified by PCR (forward: 5′-CCT CAC TCC TGG CGC GGC CGC GGG AGG TTC TGG AGG ATT GT-3′ and reverse: 5′-GGC GCC TAG AGA CGC GGC CGC GCC GCC AGA CCC TCC TCG-3′) and then inserted in place of EGFP into the Gag MLV subclone pcDNA.MLVgp.EGFP.p12 (a gift from C. Baum [ ]) opened with NotI by using the HiFi assembly kit (NEB).

Techniques: Fluorescence