The LACTB Knockout HT29 Polyclonal Cells product comprises a CRISPR/Cas9-edited polyclonal cell population derived from the HT29 human colorectal adenocarcinoma cell line, featuring targeted disruption of the LACTB gene. This polyclonal knockout model enables loss-of-function studies of LACTB, a mitochondrial serine beta-lactamase-like protein with established tumor suppressor activity. The edited population provides a heterogeneous but gene-targeted population suitable for functional genomics, signaling analysis, and phenotypic screening in a cancer-relevant epithelial background. Researchers can investigate LACTB-dependent pathways without clonal artifacts, leveraging the diversity of a polyclonal pool.
HT29 cells originate from a human female colorectal adenocarcinoma and exhibit epithelial morphology with the capacity to form polarized monolayers. This cell line harbors wild-type KRAS and a BRAF V600E mutation, representing a clinically relevant genotype found in a subset of colorectal cancers. HT29 cells are widely used as an in vitro model for studying colorectal cancer biology, including proliferation, differentiation, and response to therapeutics. The BRAF V600E mutation renders these cells particularly valuable for investigating MAPK pathway interactions with tumor suppressor mechanisms, making them an appropriate host for assessing the phenotypic consequences of LACTB knockout in a defined oncogenic context.
LACTB is a mitochondrial intermembrane space protein that functions as a lipid hydrolase, specifically catalyzing the hydrolysis of phosphatidylethanolamine (PE) into lyso-PE. This enzymatic activity is transcriptionally activated by the tumor suppressor p53, positioning LACTB as a downstream effector of p53-mediated apoptosis. Accumulated lyso-PE and alterations in mitochondrial membrane composition lead to mitochondrial outer membrane permeabilization (MOMP), promoting the release of cytochrome c and subsequent activation of caspase-9 and caspase-3 through the intrinsic apoptotic pathway. LACTB facilitates BAX and BAK pore formation, which is critical for MOMP. Additionally, LACTB interacts with the mitochondrial protein import machinery, underscoring its localized regulatory role. Disruption of LACTB abrogates p53-dependent induction of apoptosis, enhancing cell survival and proliferation. The pathway components include p53, BAX, BAK, cytochrome c, caspase-9, and caspase-3, forming a signaling axis linking lipid metabolism to programmed cell death.
In the HT29 cellular background, LACTB knockout recapitulates a loss-of-tumor-suppressor state, enabling systematic investigation of how mitochondrial lipid metabolism interfaces with oncogenic signaling. Given that HT29 cells are BRAF V600E mutant and KRAS wild-type, this model is particularly suited for dissecting p53?CLACTB interactions independent of KRAS-driven signaling. The absence of LACTB is anticipated to impair PE hydrolysis, diminish cytochrome c release, and attenuate caspase activation, thereby conferring resistance to apoptosis and promoting uncontrolled proliferation. This model holds relevance for colorectal cancer research but may also extend to breast cancer, hepatocellular carcinoma, and metabolic disorders where LACTB dysregulation has been implicated. The polyclonal nature reduces clonal selection biases, offering a more representative population for studying heterogeneous tumor cell responses.
Researchers can employ this knockout model to explore tumor suppression mechanisms, apoptosis regulation, and mitochondrial biology. Representative assays include Western blotting for LACTB and apoptosis markers (such as cleaved caspase-3), RT-qPCR to verify LACTB mRNA reduction, and cell proliferation assays (MTT or BrdU) to assess growth advantages following knockout. Apoptotic responses can be quantified by Annexin V/propidium iodide staining, and mitochondrial membrane potential changes monitored via JC-1 dye. Lipidomic profiling enables direct measurement of PE and lyso-PE levels, while colony formation assays evaluate long-term clonogenic survival. Differentiation status can be examined using markers like villin and keratin 20. This product thus supports a broad range of studies in cancer biology, lipid metabolism, and CRISPR-based functional genomics. For additional product information or technical support, please contact Ascent Research.
