The CCKBR Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from near-haploid HAP1 cells, targeting the CCKBR gene. This loss-of-function model enables investigation of cholecystokinin B receptor signaling, a GPCR responsive to gastrin and cholecystokinin. The polyclonal format provides a genetically diverse pool suitable for robust functional assays without single-cell cloning biases.
The HAP1 cell line originates from KBM-7, a chronic myeloid leukemia (CML) line from a blast crisis patient, and features a near-haploid karyotype. This genomic simplicity facilitates effective CRISPR/Cas9-mediated gene disruption, minimizing functional redundancy and enabling clear phenotypic readouts. HAP1 is widely employed for genetic screens and knockout model generation, offering a reproducible platform for studying signaling networks in a leukemia-derived context.
CCKBR is a G??q/11-coupled receptor that transduces signals from gastrin, CCK, and sulfated CCK8, activating phospholipase C ?? (PLC??) to generate IP3 and DAG. This triggers intracellular calcium release and protein kinase C (PKC) activation, which propel the MAPK/ERK cascade via MEK, leading to phosphorylation of ERK and transcription factors ELK1 and c-Fos. Concurrent PI3K/AKT pathway engagement and Src-mediated EGFR transactivation promote expression of proliferative targets like Cyclin D1 and c-Jun. Receptor modulation involves ??-arrestin, GRK, and scaffolding by PDZ proteins such as PSD-95. Upstream cytokines IL-1?? and TNF-?? further regulate CCKBR expression.
In the HAP1 background, CCKBR knockout permits precise dissection of gastrin-dependent signaling without diploid genetic complexity, directly linking pathway components to cellular outcomes. This model is instrumental for gastrointestinal cancer research, where aberrant CCKBR activity drives proliferation. The near-haploid state facilitates genome-wide synthetic lethality and drug sensitivity screens to identify therapeutic vulnerabilities resulting from receptor loss.
Applications include functional genomics, drug target validation, and high-throughput antagonist screening. Assays such as phospho-ERK western blotting, RT-qPCR for c-FOS and Cyclin D1, and calcium flux (Fluo-4) delineate signaling dynamics. Flow cytometry for cell cycle and Annexin V apoptosis, along with transwell migration, assess functional consequences. Receptor binding and RNA-seq enable pharmacological and transcriptional profiling. For technical inquiries, please contact Ascent Research.
