EEF1AKMT1 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to investigate translation elongation regulation. This product consists of a heterogeneous pool of HAP1 cells carrying targeted disruptions of the EEF1AKMT1 gene, enabling functional studies without clonal biases. The polyclonal format makes it suitable for pooled CRISPR screens and high-throughput functional genomics in a near-haploid background, providing a robust platform for dissecting the biological roles of EEF1AKMT1.
HAP1 cells are a near-haploid human cell line derived from KBM-7 chronic myeloid leukemia cells. They possess a predominantly haploid karyotype, with chromosome 8 and part of chromosome 15 diploid, allowing single-gene knockouts to yield clear phenotypes. Adherent and of male origin, HAP1 cells are extensively used in CRISPR-based functional genomics, drug target screening, and genome-wide loss-of-function studies. Their haploid nature simplifies genetic manipulation and avoids heterozygous confounding effects.
The EEF1AKMT1 gene encodes a methyltransferase that trimethylates lysine-79 of EEF1A, a translation elongation factor. This modification is regulated by mTOR signaling in response to EGF and insulin. Activated mTOR promotes EEF1AKMT1 activity, leading to elevated EEF1A methylation, enhanced global translation, and actin cytoskeleton reorganization. The enzyme uses S-adenosylmethionine as a methyl donor and directly binds EEF1A. Core pathway components include mTORC1, S6K, the eEF1 complex, and initiation factors. EEF1AKMT1 thus couples growth signals to translational output, influencing cell growth and stress responses.
In the near-haploid HAP1 background, EEF1AKMT1 knockout completely eliminates EEF1A K79 methylation, providing an unambiguous system to study the functional consequences of this modification. This model is highly relevant for cancer research, where aberrant translation drives tumorigenesis, and for neurological disorders linked to disrupted protein synthesis. The haploidy enables clean genetic screens to identify synthetic lethal partners of EEF1AKMT1. Thus, these cells serve as a powerful tool to dissect EEF1AKMT1-dependent phenotypes in growth and disease.
These polyclonal knockout cells support a wide range of research applications, including systematic dissection of the mTOR?CEEF1AKMT1?CeEF1A pathway, genome-wide CRISPR screens for modifiers of translational control, and evaluation of small-molecule inhibitors targeting protein methyltransferases. Key assays include Western blotting for EEF1A methylation, RT-qPCR for gene knockout verification, puromycin incorporation to quantify global translation rates, co-immunoprecipitation of the methyltransferase with EEF1A, and mass spectrometry for methylation site identification. Additionally, proliferation and apoptosis assays, along with RNA-seq, can be performed to assess cellular and transcriptomic outcomes. For further information, contact Ascent Research.
