The CCM2 Knockout HAP1 Polyclonal Cells are a heterogeneous population of HAP1 human near-haploid cells harboring CRISPR/Cas9-mediated disruptions of the CCM2 gene. This polyclonal knockout pool serves as a versatile loss-of-function model for studying the cellular roles of CCM2 without clonal selection, enabling researchers to assess gene disruption effects across a diverse genetic background. The product is supplied as a polyclonal population, reflecting the combined knockout profiles of multiple edited cells, and is suitable for applications ranging from pathway analysis to phenotypic screening.
The HAP1 host cell line is a fibroblast-like, near-haploid cell model originally derived from a chronic myeloid leukemia patient. Its near-haploid karyotype, with only a single copy of most chromosomes, simplifies genetic manipulation and minimizes confounding effects from heterozygous mutations, making it an ideal platform for knockout studies, genetic screens, and isogenic cell engineering. HAP1 cells retain key signaling pathways and exhibit robust proliferation, facilitating reproducible experimental workflows in cell biology and drug discovery.
CCM2 encodes an essential scaffolding protein that, together with KRIT1 (CCM1) and PDCD10 (CCM3), forms the cerebral cavernous malformation (CCM) signaling complex. This complex is critical for maintaining endothelial cell?Ccell junction integrity and negatively regulating RhoA-ROCK signaling. CCM2 facilitates the formation of a ternary complex with MEKK3 and ICAP1, promoting MEKK3-MEK5-ERK5 pathway activation to drive expression of the transcription factors KLF2 and KLF4, which are key mediators of endothelial quiescence and barrier function. Upstream inputs include VEGF, shear stress, TNF-alpha, and angiopoietin-1, while downstream targets extend to VE-cadherin, RhoA, and ROCK. Interactions with SMAD proteins further link CCM2 to transforming growth factor-beta (TGF-??) signaling. Disruption of CCM2 perturbs these networks, leading to RhoA hyperactivation, reduced KLF2/4 expression, and compromised junctional stability.
In the HAP1 near-haploid background, CCM2 knockout recapitulates core signaling defects observed in endothelial cells, offering a simplified system to dissect the molecular consequences of CCM complex loss. The absence of a second allele unmasks the full impact of CCM2 disruption on the MEKK3-ERK5 and RhoA-ROCK axes, providing a genetically clean model for mechanistic studies. This system is particularly valuable for probing protein?Cprotein interactions, assessing downstream transcriptional changes, and evaluating pharmacological interventions that target these pathways, independent of endothelial-specific factors.
This knockout model supports a wide range of research applications, including investigations into cerebral cavernous malformation pathogenesis, Rho GTPase signaling, and kinase pathway crosstalk. Representative techniques include western blotting and RT-qPCR to confirm target disruption and assess KLF2/KLF4 expression, co-immunoprecipitation to examine CCM complex assembly, RhoA pull-down activation assays, and ERK5 phosphorylation ELISA. Migration assays and RNA-seq further enable functional and transcriptomic analyses. For further information, please contact Ascent Research.
