CD28 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HAP1 near-haploid human chronic myeloid leukemia cell line, featuring targeted disruption of the CD28 gene. This model generates a loss-of-function system for the T-cell costimulatory receptor CD28, enabling pooled functional studies without clonal selection bias. These polyclonal knockout cells are designed to support high-throughput or pooled assays where genetic heterogeneity mirrors natural variation, avoiding artifacts from single-cell cloning.
The host HAP1 cell line was derived from the KBM-7 chronic myeloid leukemia line isolated from a patient in blast crisis. Its near-haploid karyotype (approximately 25 chromosomes) simplifies gene targeting because only one allele needs disruption, and its adherent growth facilitates cell-based assays. Although of myeloid origin, HAP1 cells have been widely used as a host for studying signaling pathways from various lineages, particularly when key receptors or adaptors are ectopically expressed.
CD28 is a critical costimulatory receptor that is activated by CD80/CD86 ligands, working downstream of the TCR complex (CD3, Lck, ZAP-70). Upon engagement, it recruits PI3K and Grb2, triggering the PI3K-AKT, NF-??B, and MAPK/ERK cascades. This leads to phosphorylation of AKT, GSK-3, and mTOR, and activation of transcription factors NF-??B, AP-1, and NFAT, which promote IL-2 and Bcl-xL expression, driving T-cell proliferation and survival. The receptor interacts with intracellular partners including Filamin-A and Itk, and its costimulatory signal synergizes with TCR-proximal events mediated by LAT and ZAP-70.
In HAP1 cells, CD28 knockout allows precise interrogation of costimulatory signaling in isolation, supporting studies of aberrant T-cell activation in autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. This cell model is particularly valuable for exploring the molecular basis of autoimmune disorders where CD28 signaling is dysregulated, and for evaluating immunomodulatory drugs targeting the CD28 pathway. It also serves as a platform for optimizing chimeric antigen receptor (CAR) constructs in cancer immunotherapy. The polyclonal population enhances reproducibility by minimizing clonal bias.
Research applications include flow cytometry for activation markers (CD69, CD25), IL-2 ELISA, Western blotting for phospho-AKT (Ser473) and phospho-NF-??B p65, T-cell proliferation assays (CFSE dilution), co-immunoprecipitation of CD28-PI3K complexes, and intracellular calcium flux measurements. These assays enable investigation of T-cell biology, immunotherapy development, and signal transduction. For further customization or technical consultation, please contact Ascent Research.
