The GYS1 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human colon adenocarcinoma cell line HT29. This polyclonal pool features targeted disruption of the GYS1 gene, generating a loss-of-function model for glycogen synthase 1 without clonal isolation. It serves as a robust tool for studying glycogen metabolism, cancer cell energetics, and the regulatory networks governing glycogen synthesis in an epithelial cancer background.
The HT29 host cells originate from a primary colorectal adenocarcinoma of a 44-year-old female patient and are extensively used as a model of intestinal epithelial biology and colorectal cancer. These cells display transformed metabolism, high proliferation, and dysregulated signaling, making them ideal for examining how glycogen storage contributes to tumor cell adaptation and survival. GYS1 knockout in these cells eliminates glycogen synthesis, redirecting metabolic flux.
GYS1 encodes the rate-limiting enzyme of glycogenesis, catalyzing the transfer of glucose from UDP-glucose to glycogen chains. Its activity is inhibited by phosphorylation via glycogen synthase kinase-3?? (GSK3??) and activated by dephosphorylation through protein phosphatase 1. Upstream, insulin/AKT signaling suppresses GSK3?? to promote glycogen synthesis, whereas AMPK and glucagon pathways enhance GYS1 phosphorylation to inhibit it. GYS1 interacts with glycogenin and is regulated by laforin, malin, and PPP1R3C. Disruption of GYS1 abrogates glycogen accumulation and alters glucose-6-phosphate utilization.
In colorectal cancer, metabolic reprogramming often includes glycogen accumulation to support proliferation under stress. Knocking out GYS1 in HT29 cells depletes glycogen stores and mimics metabolic defects seen in glycogen storage disease type 0, providing a model to probe how glycogen synthesis impacts tumor energetics, redox homeostasis, and drug resistance. This system uncovers the reliance of colon adenocarcinoma cells on glycogenesis downstream of oncogenic pathways such as PI3K/AKT.
These cells enable detailed investigations of glycogen synthase function via western blotting, PAS staining for glycogen, RT-qPCR, glucose uptake assays, and metabolomics. Functional assays include proliferation, colony formation, cell cycle analysis by flow cytometry, and Seahorse metabolic flux analysis to measure glycolytic and mitochondrial respiration. The model is well-suited for screening glycogen synthase inhibitors and dissecting crosstalk between insulin, AMPK, and GSK3?? in cancer metabolism. For more information, contact Ascent Research.
