The EIF2AK1 Knockout HEK293T Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout population of HEK293T cells with targeted disruption of the EIF2AK1 gene. This loss-of-function model abolishes expression of heme-regulated inhibitor (HRI) kinase, providing a powerful tool for dissecting integrated stress response signaling in the absence of endogenous HRI activity. The polyclonal format yields a heterogeneous cell pool, supporting functional studies that capture the biological variability of gene disruption.
HEK293T cells are a human embryonic kidney?Cderived line that stably expresses the SV40 large T antigen, enabling episomal replication of plasmids containing the SV40 origin. Combined with high transfection efficiency, this feature has made HEK293T a preferred host for transient and stable gene editing, as well as for biochemical and cell-based assays. The line??s robust proliferation and protein expression capacity further enhance its utility.
EIF2AK1 encodes the HRI kinase, which is activated by heme deficiency, oxidative stress, heat shock, and proteasome inhibition. Activation leads to phosphorylation of eIF2?? at serine 51, reducing global translation while increasing translation of ATF4. ATF4 transcriptionally upregulates CHOP and GADD34, which control cell fate decisions. Heme acts as a negative regulator by binding to HRI, and Hsp90 chaperones the kinase.
In HEK293T cells, EIF2AK1 knockout eliminates HRI-mediated eIF2?? phosphorylation, allowing dissection of HRI-specific functions independently of other eIF2?? kinases (PERK, PKR, GCN2). Despite being non-erythroid, HEK293T cells exhibit a robust stress response, making this knockout model ideal for studying HRI-dependent signaling in a tractable genetic background. The absence of HRI creates a clean background for examining ATF4 activation and downstream gene induction.
Applications include Western blot analysis of phospho-eIF2?? (Ser51), ATF4, and CHOP; RT-qPCR quantification of ATF4 and CHOP mRNA levels; measurement of global translation via puromycin incorporation or polysome profiling; and apoptosis assays by flow cytometry under stress conditions like heme depletion. An ATF4 luciferase reporter assay offers a functional readout for pathway activity. This polyclonal knockout pool is well-suited for high-throughput screening of stress modulators and pathway dissection. For additional details, please contact Ascent Research.
