The AKT3 Knockout HEK293T Polyclonal Cells product is a CRISPR/Cas9-edited polyclonal knockout cell population derived from HEK293T cells, designed to disrupt the human AKT3 gene. This pooled population arises from a heterogeneous mixture of edited alleles, enabling loss-of-function studies without clonal selection bias. The CRISPR/Cas9 system introduces targeted gene disruption, generating a versatile cellular model for interrogating AKT3-dependent processes in a widely utilized host background.
The parental HEK293T cell line is an immortalized human embryonic kidney epithelial line that stably expresses the SV40 large T antigen. This modification permits episomal replication of plasmids containing the SV40 origin and significantly enhances protein expression levels, making HEK293T a standard host for transient and stable transfection, viral production, and biochemical analysis. The cells exhibit robust growth and high transfection efficiency, providing an experimentally tractable system for signal transduction research.
AKT3 encodes a serine/threonine kinase that functions as a central mediator of PI3K-AKT signaling. Upon growth factor stimulation (e.g., IGF1, EGF, PDGF), PI3K generates PIP3, recruiting AKT3 to the membrane where it is phosphorylated and activated by PDK1 and mTORC2. Activated AKT3 phosphorylates multiple downstream targets: it inhibits TSC2 to relieve mTORC1 suppression, phosphorylates and inactivates FOXO1/3/4 transcription factors to reduce pro-apoptotic gene expression, and inhibits GSK3?? to promote anabolic metabolism. Additional substrates include MDM2, BAD, PRAS40, and AS160, linking AKT3 to cell survival, protein synthesis, glucose uptake, and apoptosis regulation. AKT3 interacts with PDPK1, mTORC2, HSP90, PP2A, CTMP, and TCL1A, which fine-tune its localization and activity. The kinase is particularly important in neuronal development, and its dysregulation is implicated in glioblastoma, melanoma, and overgrowth syndromes.
Loss of AKT3 function in HEK293T cells provides a clean background to dissect isoform-specific contributions of AKT family members in oncogenic signaling, metabolism, and apoptosis. Given HEK293T??s ease of manipulation, the knockout cells can be complemented with mutant or isoform-specific AKT variants, enabling structure-function analyses and precise mapping of phosphorylation-dependent interactions. This genetic deletion model eliminates confounding endogenous AKT3 activity, thereby increasing the signal-to-noise ratio in phospho-specific detection assays and expanding the dynamic range for inhibitor response studies.
This knockout cell population is well-suited for dissecting PI3K-AKT pathway mechanisms in cancer and neurobiology. Common applications include Western blotting and RT-qPCR to confirm AKT3 depletion, MTT and CFSE proliferation assays, apoptosis detection via Annexin V/PI flow cytometry, and phospho-specific flow cytometry to monitor pathway activity. The HEK293T background facilitates inhibitor screening and complementation studies with exogenous AKT3 variants. For further information or to request technical support, please contact Ascent Research.
