Biotinylation of nearby tumor suppressor proteins and the effect of deleting the exon of the mitochondrial calcium channel protein

Cancer is a leading cause of mortality characterized by uncontrolled cell growth. Neuroblastoma, a type of malignant tumor affecting the nervous system, originates from immature nerve cells and is studied using the SH-SY5Y cell line derived from a child's bone marrow. This cell line along with HEK293T is crucial for neuroscience and cancer studies. Mitochondria, the cell's powerhouse, contain proteins like MICU1 and MICU2 that regulate calcium ion influx, playing a significant role in cellular processes. Key techniques include Biotin Identification and Ascorbate Peroxidase 2 for biotinylation of neighboring proteins and CRISPR/Cas9 for genetic modifications. Studies have shown that deletions in exon three reduce mitochondrial calcium ion uptake without inducing apoptosis and when Ascorbate Peroxidase 2 is fused with the IDG_CACNA2D3_OE_1 vector, it allows for the biotinylation of tumor suppressor proteins like P53, which are crucial for initiating apoptosis. This project investigates the impact of deleting exon three of the mitochondrial calcium channel protein and explores potential biotinylation of tumor suppressor calcium channel proteins using Ascorbate Peroxidase 2. The deletion involved cloning sgRNAs into Lenti CRISPR V2 Puro and the pSp Cas9 (BB) – 2A -GFP (PX 458) vectors, followed by transfection into SH-SY5Y cells. Although Nanodrop analysis indicated contamination affecting sample purity, Sanger sequencing confirmed the correct insertion location. RNA extraction yielded acceptable results, while RT-qPCR indicated slight redundancy due to pipetting errors or unstable housekeeping genes. The IDG_CACNA2D3_OE_1 and APEX2-OMM vectors were utilized in biotinylation, with codon optimization of the IDG_CACNA2D3_OE_1, yielding three successful codon optimimzed samples out of 24. Not all 24 samples were codon optimized due to incomplete Kinase, Ligase, and DpnI incubation time, resulting in wild-type sequences outcompeting mutants. Gradient PCR for APEX2-OMM vector yielded optimal results. Ultimately, the experiment was successful, with future studies recommended, focusing on minimizing troubleshooting for improved outcomes.