The BAD Knockout Ca Ski Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal population of Ca Ski human cervical carcinoma cells with targeted disruption of the BAD gene. This engineered model enables the study of loss-of-function effects for the pro-apoptotic BH3-only protein BAD in a well-characterized, HPV-16-positive epithelial background. The polyclonal format provides a heterogeneous knockout cell pool that reflects the diversity of editing outcomes produced by the CRISPR/Cas9 system, offering a robust tool for functional genomics, drug discovery, and apoptosis research without clonal selection biases.
Ca Ski cells were originally derived from a metastatic site of a cervical squamous cell carcinoma and harbor integrated human papillomavirus type 16 (HPV-16) genomes. As an adherent epithelial cell line, Ca Ski is widely employed to model cervical carcinogenesis, HPV-driven oncogenesis, and intrinsic apoptosis regulation. The presence of HPV oncoproteins E6 and E7 in these cells perturbs p53 and retinoblastoma protein pathways, creating a unique context in which to interrogate mitochondrial apoptosis signaling and survival mechanisms relevant to cervical cancer progression.
BAD (BCL-2 antagonist of cell death) functions as a pro-apoptotic BH3-only member of the BCL-2 family, acting as a stress sensor that promotes programmed cell death. BAD heterodimerizes with anti-apoptotic proteins BCL-2, BCL-XL, and BCL-W, thereby liberating the effector proteins BAX and BAK to permeabilize the outer mitochondrial membrane. This event triggers cytochrome c release, APAF-1-mediated apoptosome assembly, and sequential activation of caspases-9 and -3. BAD activity is tightly controlled by upstream survival kinases: AKT, PKA, RSK, and p70S6K phosphorylate BAD at conserved serines (including Ser112, Ser136, and Ser155), promoting its sequestration by 14-3-3 adaptor proteins and subsequent inactivation. Conversely, protein phosphatase 2A (PP2A) dephosphorylates BAD, restoring its pro-apoptotic function. The RAS/ERK and PI3K/AKT pathways, activated by growth factors such as IGF-1 and IL-3, converge on BAD to regulate cell survival.
In the Ca Ski cervical cancer background, disruption of BAD is expected to reduce mitochondrial apoptotic priming, conferring a survival advantage and resistance to apoptotic stimuli. Given the role of HPV in evading apoptosis, BAD knockout in this model provides insight into the interplay between viral oncoproteins and intrinsic cell death circuitry. Researchers can use this system to dissect how phosphorylation-dependent regulation of BAD by AKT and ERK integrates with HPV-mediated signaling, and to explore the contribution of BAD loss to chemoresistance, a hallmark of cervical cancer.
Typical applications include western blot analysis of BAD, cleaved caspase-3, and phospho-BAD isoforms to dissect signaling events; Annexin V/propidium iodide staining to quantify apoptosis; JC-1 assays to assess mitochondrial membrane potential; co-immunoprecipitation for BCL-2 family interactions; and cisplatin or other chemotherapeutic sensitivity profiling using MTT-based viability assays. The polyclonal pool is also suited for siRNA or inhibitor screens targeting the PI3K/AKT and RAS/ERK axes, or for evaluating BCL-2 family inhibitors in an apoptosis-compromised environment. For additional details, please contact Ascent Research.
