BBC3 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population targeting the BBC3 gene in the HT29 human colorectal adenocarcinoma cell line. This product provides a loss-of-function model in which BBC3, encoding the pro-apoptotic BH3-only protein PUMA, is disrupted using CRISPR/Cas9-mediated gene editing, generating a heterogeneous pool of cells with targeted genomic modifications. The polyclonal format allows researchers to study the collective impact of various knockout alleles, avoiding clonal selection biases while enabling robust functional analysis in a well-characterized epithelial background.
The HT29 host cell line was originally isolated from a 44-year-old female with colorectal adenocarcinoma and exhibits an adherent epithelial morphology. These cells express characteristic colon epithelial markers such as cytokeratin 20 (CK20) and mucin 2 (MUC2) and retain the potential for enterocytic differentiation. HT29 cells are widely used as a model of intestinal epithelial biology, including investigations of colon cancer pathogenesis, drug transport mechanisms, and epithelial barrier integrity, making them an ideal platform for dissecting apoptosis-related signaling in a therapeutically relevant context.
PUMA is a critical BH3-only member of the BCL2 protein family that functions as a key sensor of cellular stress. Under conditions such as DNA damage or endoplasmic reticulum (ER) stress, PUMA expression is transcriptionally upregulated by factors including p53, E2F1, FOXO3a, p73, and CHOP. Once induced, PUMA binds and neutralizes anti-apoptotic BCL2 family proteins??BCL2, BCL-XL, and MCL1??thereby releasing BAX and BAK. This facilitates BAX/BAK oligomerization at the mitochondrial outer membrane, triggering mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and subsequent formation of the APAF1/caspase-9 apoptosome. The resulting activation of effector caspases such as caspase-3 executes the apoptotic program. Direct interactions between PUMA and BCL2, BCL-XL, and MCL1 have been demonstrated, and its function is integrated into the p53-dependent intrinsic apoptotic pathway.
In the HT29 colorectal adenocarcinoma background, the disruption of PUMA provides a valuable tool for examining how pro-apoptotic signaling interfaces with cancer cell survival mechanisms. Colorectal cancers frequently exhibit dysregulation of the p53 pathway, and PUMA is a principal mediator of p53-dependent apoptosis following genotoxic insults. By comparing wild-type and PUMA-knockout HT29 polyclonal populations, researchers can delineate the contributions of BH3-only proteins to chemotherapeutic sensitivity, DNA damage responses, and the balance between pro-survival and pro-death signals. This model is particularly relevant for understanding resistance mechanisms in colon cancer and for identifying strategies to reactivate apoptosis via BH3 mimetics or other modalities.
Typical experimental applications include analyzing p53-mediated apoptosis after treatment with DNA-damaging agents, evaluating the role of BH3-only proteins in the intrinsic apoptotic cascade, and screening for modulators that bypass PUMA deficiency. Representative assays include Western blotting for PUMA, p53, and cleaved caspase-3; real-time RT-qPCR to quantify BBC3 mRNA; flow cytometry using Annexin V and propidium iodide staining to measure apoptosis; caspase-3/9 activity assays; co-immunoprecipitation to probe BCL2 family interactions; cell viability assays such as MTT or CellTiter-Glo following genotoxic stress; and p53 transcriptional reporter assays. For additional product information or to discuss your experimental requirements, please contact Ascent Research.
