Constructing an inducible adenovirus system for long non-coding RNA knockdown
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Citation
Abstract
Sickle cell disease (SCD) is an inherited monogenic hemoglobinopathy that affects millions of people worldwide and has been recognized as a global public health problem by the World Health Organization. When sickle red blood cells are damaged and misshaped they tend to block blood flow which can lead to acute vaso-occlusive episodes, hemolytic anemia, stroke, eye retinopathy, and infections. An increase in fetal hemoglobin (HbF) expression can diminish the severity of SCD because HbF can inhibit the polymerization of deoxy-sickle hemoglobin (HbS). In a previous study conducted by the lab, researchers constructed a lentivirus vector to knockdown HBS1L-MYB-intergenic region long non-coding RNA (HMI-lncRNA), which would activate HBG (HBG1 and HBG2) gene expression ultimately increasing HbF synthesis.1 To better understand the role of HMI-lncRNA in regulating HBG, we wanted to have an inducible knockdown system so that we can control the activation and silencing of HMI-lncRNA expression at different time points or development statuses.2 Due to lentivirus vectors having a larger packaging capacity, it gives them a disadvantage in efficient gene delivery compared to adenovirus vectors.3 Therefore, we designed an improved vector to knockdown HMI-lncRNA. This adenovirus vector has a tetracycline-controlled Tet-on/off system incorporated into the short hairpin RNA interference construction. It also carries a green fluorescent protein for selecting and tracking the cells with HMI-lncRNA knockdown. This new inducible adenovirus knockdown system would be a useful tool for our HMI-lncRNA study as well as other lncRNA function studies in other cell lineages.