Associate Professor University of Utah, United States
Introduction:
Introduction: Intervertebral disc (IVD) disease and associated back pain is a major healthcare concern in the US with health care costs exceeding $100 billion annually. Degeneration of the IVD has been closely associated with decreased cell activity and senescence, with studies indicating that up to 80% of cells within a severely degenerated disc demonstrate a senescent state. Recently in a genome wide CRISPRa screen, our lab discovered a novel zinc finger protein, ZNF865, that produced robust cell-engineering phenotypes relating to cell cycle, protein processing and cellular senescence. Preliminary data suggests that this gene is differentially regulated in degenerative IVDs. One of the most common models of IVD disease is in the rat, however, all studies on this gene have been performed in human immortalized and primary samples. This indicates a critical need to identify orthologs within other species to allow for useful animal models to be developed. Here we investigate the phenotype and functional outcomes of CRISPR regulation (CRISPRi) of ZNF865 rat nucleus pulposus cells to determine if the effects that are present within human experiments are maintained and evaluate whether this ortholog can be used to appropriately model ZNF865 regulation in rats.
Materials and
Methods: Materials and
Methods: Rat nucleus pulposus cells obtained from the caudal IVD of sacked animals were isolated by digestion with collagenase and pronase and plated onto tissue culture plastic. Following isolation, cells were transduced with CRISPRi lentiviral vectors (1A) targeting the rat ZNF865 (G1, G2 or G3) and non-target controls (NTC). Fold change gene expression was measured using qRT-PCR of RNA extracted from ZNF865 downregulated and NTC cells 1 week after transduction (n=4-6). Cell proliferation was evaluated by imaging and fluorescent cell counting for 1 week following successful transduction (n=4-6). Senescence was evaluated using SA-β-gal staining 10 days following successful transduction (n=4).
Results, Conclusions, and Discussions:
Results: Fold change gene expression showed significant repression by KRAB in all the tested ZNF865 guides (1B). Downregulation of ZNF865 showed a significant decrease in cell proliferation in each of the three ZNF865 guides that were tested compared to the NTC (1C) with doubling times increasing by 196 ± 49%, 144 ± 18% and 175 ± 16% in G1, G2 and G3 respectively compared to the NTC (1D). Downregulation of ZNF865 also lead to increased cellular senescence in all ZNF865 guides compared to NTC (1E,1F). One-way (qRT-PCR, doubling time, SA-β-gal) and two-way ANOVA (proliferation) were used to determine significance ( = 0.05). iscussion: This research examines the effects of CRISPR-guided gene modulation of ZNF865 in rat nucleus pulposus cells in vitro. Overall, our results suggest ZNF865 in the rat regulates key processes relating to cell activity and senescence. Upon downregulation of ZNF865, significant decreases in cell proliferation was seen, with increased levels of senescence occurring compared to the nontarget. These results indicate that the rat ZNF865 gene is necessary for the normal function of these cells and provides evidence that the function of this gene is conserved across the human and rat genome. Overall, this work establishes the rat as an acceptable animal model to further study ZNF865 within the intervertebral disc.
