Browsing by Subject "Gene knockdown"

Severe acute respiratory syndrome coronavirus 2 is the pathogen behind COVID-19, a contagious disease that has caused millions of deaths since the beginning of the global pandemic in 2019. It is essential to gain more knowledge about the factors affecting viral entry and infection to understand prevention and treatment possibilities for the pathogen. Initially, an immunostaining assay was set up and showed that at eight hours post-infection, the virus had entered the target cells and started protein production. Five host genes of interest, identified by collaborators, were transiently silenced using small interfering RNA knockdown of cell lines. Once the silencing had been optimized, the effect on SARS-CoV-2 infection was monitored at eight and 24 h post-infection. The main pipeline used to follow the cell entry and replication was an extraction of total RNA, reverse transcription reaction followed by quantitative digital droplet polymerase chain reaction, showing the concentrations of the expressed target genes in the cells. The protocols of the transfection and ddPCR were tested and optimized to provide reproducible and reliable results. Out of six tested transfection reagents, DharmaFECT 2 showed the most effective results, decreasing the expression of target genes DDX3X and PDE4A by more than 75% after a 24 h incubation. The produced knockdown cells were infected with a produced and purified stock of SARS-CoV-2. Plaque assays were made from samples collected 24 hours post-infection, but no difference could be seen in the titer of the virus between the transfected cells and negative control cells. In conclusion, using digital droplet polymerase chain reaction to follow the effect of host gene knockdown on virus replication is possible as long as the initial gene expression is high enough.