scrambled sgrna control  (Applied Biological Materials Inc)

Journal: Journal of Virology

Article Title: Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7

doi: 10.1128/jvi.01178-24

Figure Lengend Snippet: CTD serine 2, but not serine 7, is required for efficient viral gene expression. HEK-293T cells were co-transfected with a construct expressing a sgRNA transcribed by Pol III as a transfection control and plasmids expressing amanitin-resistant RPB1 with wild-type (WT) or indicated mutant CTDs, or the control vector pcDNA3 lacking any polymerase. The next day, α-amanitin was added to degrade endogenous RPB1 for 24 h followed by infection with HSV-1 strain 17+, and viral gene expression was measured by RT-qPCR at 24 hpi. Means of three biological replicates with standard error are plotted. Statistically significant differences to mock are indicated as * P < 0.05, ** P < 0.01, *** P < 0.001. S2A: serine 2 to alanine, S7A: serine 7 to alanine, S7E: serine 7 to glutamate.

Article Snippet: For qPCR analysis, the assay was repeated but scaled up to 24-well plates, this time using 50 ng of the vector pU6.scr_sgRNA in place of Azurite, which expresses a scrambled control sgRNA based on Addgene #62285 from the RNA Pol III promoter U6.

Techniques: Gene Expression, Transfection, Construct, Expressing, Control, Mutagenesis, Plasmid Preparation, Infection, Quantitative RT-PCR