pfugwmcherry lentivirus vector  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: RNA-targeted proteomics identifies YBX1 as critical for efficient HCMV mRNA translation.

doi: 10.1073/pnas.2421155122

Figure Lengend Snippet: Fig. 4. YBX1 regulates translation of HCMV immediate early transcripts. (A) CRISPR was used on hTERT-MRC5 fibroblasts to generate either a control cell line (NT, black) or a YBX1-depleted cell line (ΔYBX1 −Dox, white). YBX1 was recovered in knockdown cells with a lentivirus expressing YBX1 under a dox inducible promoter (ΔYBX1 +Dox, gray). Cells were infected with HCMV at an MOI of 3 and harvested at 24 hpi. Cytoplasmic lysates resolved over a 10 to 50% linear sucrose gradient and RNA analyzed via qRT-PCR. (B) Translation efficiency was calculated by comparing the abundance of RNA transcripts with polysomes to monosomes. (C) Cells were infected as in (B) and harvested for protein analysis via western blot. (n = 3 ± SEM, *P < 0.05, **P < 0.01).

Article Snippet: For gRNAs, a lentivirus vector was purchased from Addgene (Paw13.lentiguide.mCherry #104375) and PCR amplified with primers “pAW13 F” and “pAW13 R.” Multiple gRNAs were designed with a 30 nucleotide sequence to specifically target the MIE 5’ UTR or scrambled sequence, ordered as gblocks to contain homology to the amplified lentivirus backbone, and assembled using Gibson Assembly.

Techniques: CRISPR, Control, Knockdown, Expressing, Infection, Quantitative RT-PCR, Western Blot