Intro: Hypovitaminosis D (vitamin D deficiency) has been observed in ageing patients with brain calcification and loss of the vitamin D receptor leads to abnormal calcification of the basal ganglia and thalamus. We have found that vitamin D can reverse calcification of human osteosarcoma SaOs-2 cells in vitro, in apparent contrast to its known effects of increasing bone strength in patients with Rickets and Osteomalacia. Vitamin-D functions through binding to two Vitamin-D responsive proteins; the vitamin D receptor (VDR) and Protein Disulfide isomerase A3 (PDIA3). The aim of this project was to establish VDR and PDIA3 knockout SaOs-2 cells using CRISPR-Cas9 technology. Methods: We designed guide RNA (gRNA) sequences against PDIA3 and VDR using ChopChop, selecting only gRNAs with low predicting non-specific binding probabilities. These gRNA sequences were ordered as oligonucleotides and dimerised before directional cloning into a Cas-9 plasmid. Plasmids were amplified in DH5 E. coli and purified before transfection into SaOs-2 cells together with a plasmid containing the puromycin resistance gene. Cells were treated with puromycin (1 ug/ml) for 4 days to eliminate non-transfected cells. SaOs-2 cells were maintained for 7 days before being passaged and plated for colony selection. Results: Real Time quantitative PCR showed 1 SaOs-2 clone had non-detectable levels of PDIA3 while 4 out of 6 clones had no detectable VDR mRNA relative to wild type cells. Two clones were selected for further analysis. Western blotting of these two clones probing for VDR and PDIA3 confirmed there were no detectable levels of these two proteins. Conclusion: We successfully knocked out expression of the Vitamin-D receptors VDR and PDIA3 in SaOS2 cells. These cells will be used for further study of Vitamin-D related signaling.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-1657 |
| Date | 15 May 2020 |
| Creators | Pyburn, Jaeden, Keasey, Matthew |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Appalachian Student Research Forum |
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