The DOC2A Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human embryonic kidney HEK293T cell line, engineered to disrupt the DOC2A gene. This polyclonal knockout model offers a robust tool for investigating DOC2A-mediated calcium-dependent exocytosis and neurotransmitter release mechanisms. The heterogeneous pool of edited cells provides a versatile system for loss-of-function studies without the limitations of clonal selection, enabling efficient screening and biochemical analyses in a well-characterized cellular background.
HEK293T cells are a widely used derivative of the HEK293 line, stably expressing the SV40 large T antigen, which drives high-level episomal replication of plasmids containing the SV40 origin of replication. This feature makes HEK293T an excellent host for transient protein expression and viral production, facilitating downstream applications such as reporter assays, protein interaction studies, and reconstitution experiments. The cell line’s robust growth characteristics and ease of transfection ensure reproducible results in both small-scale and high-throughput formats.
DOC2A (Double C2 Domain Alpha) is a calcium sensor that promotes SNARE complex assembly essential for synaptic vesicle priming and fusion. It bridges vesicles to the plasma membrane through interactions with Munc13-1 and the SNAREs Syntaxin-1A, SNAP-25, and Synaptobrevin-2. DOC2A functions downstream of Ca2+ influx and upstream of neurotransmitter release, with regulatory input from Munc18-1 and Synaptotagmin-1. As a component of the synaptic vesicle cycle and calcium signaling pathways, DOC2A loss impairs efficient vesicle docking and exocytosis, positioning it as a central regulator of neuronal communication.
In the HEK293T background, this knockout model simplifies dissection of DOC2A’s molecular interactions and exocytic functions. Despite its non-neuronal origin, HEK293T expresses secretory machinery and can be engineered to reconstitute regulated secretion, enabling targeted studies of DOC2A-dependent pathways. The polyclonal population captures diverse editing outcomes, allowing identification of consistent phenotypes and minimizing clonal artifacts??ideal for validating protein?Cprotein interactions and signaling cascades. Researchers can isolate DOC2A-specific effects on vesicle priming and fusion without interference from neuronal networks.
These DOC2A knockout cells are suitable for a range of research applications including CRISPR knockout screening, calcium-dependent secretion assays, and neurotransmitter release studies using techniques such as FM dye imaging. Protein interaction analyses via co-immunoprecipitation with SNARE complex components (e.g., Syntaxin-1A, SNAP-25) and calcium imaging experiments are readily supported. Typical validation assays include Western blotting to confirm DOC2A ablation, RT-qPCR for transcript-level assessment, and immunofluorescence to monitor subcellular localization changes. For further information, please contact Ascent Research.
