The AACS Knockout HT29 Polyclonal Cells product comprises a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HT29 human colorectal adenocarcinoma cell line, designed for loss-of-function studies of the AACS gene. This polyclonal pool carries a CRISPR/Cas9-mediated gene disruption, ensuring a heterogeneous knockout background that models the genetic variability often observed in tumor cell populations. The knockout targets acetoacetyl-CoA synthetase (AACS), an enzyme that catalyzes the ATP-dependent ligation of acetoacetate to acetoacetyl-CoA, a critical entry point for ketone body carbon into lipid and cholesterol biosynthetic pathways. The ready-to-use format supports robust experimental workflows in cancer metabolism, metabolic signaling, and drug sensitivity research without the need for single-cell cloning.
HT29 is a mucin-producing human colon carcinoma cell line originally isolated from a 44-year-old Caucasian female with colorectal adenocarcinoma. This cell line is widely employed as an in vitro model for intestinal epithelial biology, drug absorption, and cancer research, owing to its capacity to differentiate into enterocyte-like cells under appropriate culture conditions. HT29 cells exhibit endogenous lipid metabolism activity and express key components of the mevalonate pathway, making them particularly suitable for dissecting the interplay between ketone body utilization, de novo lipogenesis, and cholesterol biosynthesis in a colorectal cancer context.
AACS functions at the crossroads of ketone body catabolism and anabolic lipid metabolism. It converts acetoacetate, derived from ketogenic diets or fasting states, into acetoacetyl-CoA, which then feeds into the HMGCS1-mediated synthesis of HMG-CoA, the substrate for the rate-limiting cholesterol biosynthetic enzyme HMGCR. Downstream of HMGCR, the mevalonate pathway produces isoprenoids and cholesterol, while acetoacetyl-CoA also provides carbon for fatty acid synthesis via FASN. AACS expression is transcriptionally regulated by PPAR??, insulin, glucagon, ketone bodies, and SREBP1, integrating systemic metabolic cues. Its activity directly impacts the cellular pools of acetoacetyl-CoA, HMG-CoA, mevalonate, and downstream lipids, positioning AACS as a metabolic hub linking nutrient availability to membrane biogenesis and signaling lipid synthesis in cancer cells.
In the HT29 colorectal adenocarcinoma background, AACS disruption is expected to impair the utilization of acetoacetate for lipid and cholesterol synthesis, thereby compromising metabolic flexibility under conditions of nutrient stress or ketone body abundance. This knockout model enables the investigation of ketone-dependent lipogenic pathways that may support tumor cell proliferation, survival, and statin resistance. By abrogating AACS function, researchers can dissect the contribution of ketone body-derived carbon to colorectal cancer cell lipidomes and assess whether AACS-dependent cholesterol synthesis promotes membrane integrity and signaling in the tumor microenvironment. The polyclonal nature of the knockout population further recapitulates intratumoral heterogeneity, offering a physiologically relevant system for evaluating metabolic vulnerabilities.
This product is suited for a wide range of experimental applications, including the assessment of ketone body utilization in intestinal epithelial cells, metabolic reprogramming in colorectal cancer, and the cross-talk between dietary fat or ketogenic interventions and tumor lipid metabolism. Typical assays include cellular lipid synthesis measurements via 14C-acetate incorporation, cholesterol quantification with Amplex Red, Seahorse metabolic flux analysis, and statin dose-response curves. Additional readouts such as western blotting for AACS pathway components (e.g., HMGCR, FASN), RT-qPCR profiling, and RNA-seq-based transcriptomics allow comprehensive mechanistic dissection. The AACS Knockout HT29 Polyclonal Cells serve as a powerful tool for identifying metabolic liabilities and evaluating therapeutic strategies targeting the mevalonate pathway in colorectal cancer. For further information, please contact Ascent Research.
