Genetics & Molecular Biology

CRISPR & Gene Perturbation

I have generated multiple CRISPR-edited iPSC cell lines (Parkin KO, α-synuclein KO, LRRK2 KO, GBA KO) to assess protein function and mimic genetic forms of neurodegenerative diseases. GBA knockout heightened vulnerability to fibril-induced inclusions, whereas α-synuclein knockout abolished pathology entirely. Validation includes PCR, Western blotting, karyotyping, and ICC for pluripotency markers.

RNA Interference & Oligonucleotide Biology

I design, optimize, and validate siRNA, shRNA, ASO, and SSO-mediated knockdown assays in iPSC-derived neurons and cancer cell lines.

Delivery methods include gymnotic uptake (media addition), lipid-based transfection, and electroporation/nucleofection. I integrate RT-qPCR, protein-level (Western/ELISA), and functional neuronal readouts to assess potency, durability, specificity, and mechanism-of-action.

I have miniaturized assays into 96/384-well formats with SOP and repeatability metrics for scalable, screening-ready execution, translating RNAi results into clear go/no-go and target-prioritization recommendations for preclinical programs.

Molecular Cloning & Construct Design

End-to-end cloning workflows including restriction cloning, Gibson assembly, Golden Gate cloning, PCR/primer design, bacterial transformation, colony screening, plasmid preparation (MiniPrep/MaxiPrep), Sanger sequencing, and endotoxin-free DNA prep. I design constructs for membrane-associated and endolysosomal proteins, with expression verification by flow cytometry and/or immunoassay. Stable cell line generation uses selection markers (Puromycin, G418, Blasticidin) with single-cell cloning and pool evaluation.

Viral Transduction & Expression

I produce and optimize lentiviral, adenoviral, and AAV/rAAV constructs for transduction in iPSC-derived neurons and mammalian cell lines. Key applications include: (1) lentiviral shRNA knockdown of LAMP2, which I demonstrated is sufficient to drive α-synuclein inclusion formation even without immune challenge — a critical finding from my Nature Neuroscience 2024 paper; (2) adenoviral overexpression of α-synuclein-HA to show that endogenous protein is actively recruited into pathological inclusions; and (3) reporter construct expression for live-cell tracking. I handle full vector design (cassettes, promoters, tags), MOI optimization, titering, and biosafety compliance across BSL-2 workflows.

Lentiviral shRNA-mediated LAMP2 knockdown in iPSC-derived DA neurons. Western blot confirms >80% reduction in LAMP2 protein. Confocal shows that LAMP2 loss alone — without immune challenge — is sufficient to induce α-synuclein inclusion formation, establishing lysosomal dysfunction as the critical vulnerability in Lewy body pathogenesis. From Bayati et al., Nature Neuroscience 2024.

Adenoviral overexpression of α-synuclein-HA in iPSC-derived DA neurons. Phospho-synuclein (pSyn S129) accumulation is dramatically enhanced under dual-hit conditions (PFF + IFN-γ), confirming that endogenous α-synuclein is recruited into pathological inclusions. Western blot quantification shows dose-dependent pSyn increase. Bayati et al., Nature Neuroscience 2024.

Biochemical fractionation of α-synuclein inclusions from iPSC-derived DA neurons. Sequential extraction (Triton-soluble → SDS-soluble → pellet) reveals enrichment of α-synuclein and HDAC6 (an aggresome marker) in the insoluble pellet fraction under dual-hit conditions, confirming the formation of detergent-resistant aggregates. Bayati et al., Nature Neuroscience 2024.

Lysosomal Biology & Protein Biochemistry

My work on lysosomal biology spans from organelle-level mechanistic studies to biochemical characterization. I showed that IFN-γ specifically downregulates LAMP1, LAMP2, TFEB, and NRF2 in DA neurons — but not cortical neurons — creating a selective vulnerability window for Lewy body formation (Nature Neuroscience 2024). I use galectin-3 as a reporter for lysosomal membrane permeabilization, revealing that fibrils rupture lysosomes and escape into the cytoplasm. My Lyso-IP approach (HA-tagged TMEM192-RFP + magnetic bead immunoprecipitation) confirmed PFF arrival at lysosomes within 2 minutes of uptake.

For protein biochemistry, I perform recombinant α-synuclein purification (Ni-NTA affinity), preformed fibril (PFF) generation (37°C shaking, 5 days, sonication), size exclusion chromatography (SEC), and DLS particle sizing. I developed a custom nanogold-PFF conjugation protocol — published in STAR Protocols (2023) as first author — enabling direct EM visualization of fibril trafficking without relying on antibody specificity.