The KLHL35 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the KLHL35 gene. This loss-of-function model is generated in the HEK293T human embryonic kidney epithelial cell line and provides a powerful tool for dissecting the biological roles of KLHL35 in the ubiquitin-proteasome system. The polyclonal nature of the knockout population captures a range of genetic disruptions, enabling robust functional studies without the biases associated with single clonal isolates. Researchers can utilize these cells to investigate the molecular consequences of KLHL35 ablation on protein homeostasis and downstream cellular processes.
The host cell line, HEK293T, is a highly transfectable derivative of HEK293 cells, stably expressing the SV40 large T-antigen. This feature facilitates efficient episomal replication of plasmids containing the SV40 origin of replication, making HEK293T cells a preferred system for transient protein expression, viral production, and a wide range of cell-based assays. Their well-characterized epithelial morphology and robust growth characteristics ensure reproducibility in experimental workflows.
At the molecular level, KLHL35 functions as a substrate adaptor for the Cullin3 (CUL3)-RING-box protein 1 (RBX1) E3 ubiquitin ligase complex. It recruits specific target proteins to the CUL3-RBX1 core, enabling their ubiquitination by E2 ubiquitin-conjugating enzymes and subsequent degradation by the 26S proteasome. Through this mechanism, KLHL35 regulates the abundance of effector proteins involved in cell cycle progression, apoptosis, and cytoskeleton organization. Although direct substrates of KLHL35 are largely unknown, its interaction with core components of the ubiquitin-proteasome machinery positions it as a critical node in protein quality control and signal-dependent proteolysis.
In the HEK293T background, this knockout model enables systematic exploration of KLHL35-dependent ubiquitination pathways. Because HEK293T cells possess an intact ubiquitin-proteasome system and are amenable to facile genetic manipulation, they provide an ideal setting for comparing global ubiquitination profiles between wild-type and KLHL35-disrupted populations. Loss of KLHL35 may lead to stabilization of its putative substrates, offering a means to identify and validate novel targets through differential proteomic or biochemical approaches. The model thus serves as a foundational platform for uncovering the physiological substrates of KLHL35 and understanding how their accumulation impacts cellular fitness.
Typical applications include RT-qPCR and Western blotting for quantifying KLHL35 transcript and protein levels. Functional assays such as ubiquitination and proteasomal degradation assays assess the consequences of KLHL35 loss on substrate turnover. Immunofluorescence studies can reveal changes in subcellular localization of potential targets, while cell viability and proliferation assays provide phenotypic insights. RNA-seq enables transcriptome-wide profiling of gene expression alterations. These cells are suitable for disease modeling of ubiquitin-related disorders and for screening regulators of CUL3-based ligases. For further details, contact Ascent Research.
