The C2CD2L Knockout HAP1 Polyclonal Cells consist of a polyclonal population of human HAP1 cells subjected to CRISPR/Cas9-mediated gene disruption of C2CD2L. This polyclonal knockout format yields a heterogeneous pool of loss-of-function alleles, ensuring experimental robustness and reproducibility. The product is designed for researchers investigating the role of C2CD2L in insulin secretion and exocytosis, providing a versatile tool for functional genomics and pathway analysis.
The host cell line, HAP1, is a human near-haploid myeloid leukemia cell line established from KBM-7 chronic myeloid leukemia cells. HAP1 cells are adherent, male (XY), and maintain a near-haploid karyotype, which simplifies genetic manipulation. Originally a model for leukemia, HAP1 has become a preferred platform for CRISPR-based functional screens due to the ease of achieving single-allele disruption, eliminating the need for homozygous knockout in polyclonal populations.
C2CD2L encodes an endoplasmic reticulum?Cplasma membrane (ER-PM) tethering protein that functions as a calcium-dependent lipid transfer protein. In pancreatic beta cells, C2CD2L facilitates insulin granule exocytosis by modulating plasma membrane phosphatidylinositol 4-phosphate (PI4P) levels and directly interacting with the SNARE complex components syntaxin-1A, SNAP-25, and VAMP2. Glucose stimulation triggers a cAMP/PKA signaling cascade, leading to PKA-mediated phosphorylation of C2CD2L, which enhances granule docking and fusion. The protein operates downstream of key insulin secretion regulators including the glucose transporter GLUT2, glucokinase, ATP-sensitive potassium (KATP) channels, and voltage-gated calcium channels, and also integrates diacylglycerol signals, thereby coupling metabolic states to exocytotic output.
Although HAP1 cells lack the native insulin secretion machinery of pancreatic beta cells, the C2CD2L knockout in this near-haploid background offers a pristine genetic environment to dissect fundamental mechanisms of ER-PM contact site function and exocytosis. The polyclonal population circumvents clonal artifacts and is well-suited for examining conserved processes such as lipid transfer and SNARE-mediated membrane fusion. Additionally, the haploid genome facilitates secondary genetic manipulations for modifier screens, making this model a powerful resource for identifying regulators of C2CD2L-dependent pathways.
This polyclonal knockout cell pool is applicable to a broad range of experimental approaches, including insulin secretion mechanism studies, diabetes drug screening, and exocytosis pathway analysis. Researchers can employ western blotting, RT-qPCR, and Sanger sequencing to validate target disruption, immunofluorescence to assess protein localization, and co-immunoprecipitation to probe interactions with syntaxin-1A or SNAP-25. Functional assays such as glucose-stimulated insulin secretion, when combined with ectopic expression of missing components, or calcium imaging enable detailed mechanistic investigations. For further details, please contact Ascent Research.
