The HLTF Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population of HEK293T cells with targeted disruption of the HLTF gene. This polyclonal model provides a genetically heterogeneous loss-of-function system suitable for pooled functional screens and mechanistic studies of HLTF-dependent processes in a human epithelial background.
HEK293T is an immortalized human embryonic kidney epithelial cell line stably expressing SV40 large T antigen, enabling high-level episomal replication of SV40 origin-containing plasmids. These cells are widely adopted for their robust growth, high transfectability, and compatibility with a broad range of assays, including DNA damage response analyses. Their epithelial origin and transformed phenotype render them a practical platform for investigating cancer-relevant signaling pathways.
The HLTF gene encodes a SWI/SNF-related chromatin remodeling ATPase that is pivotal in DNA damage tolerance and tumor suppression. HLTF functions downstream of p53 and ATM/ATR kinases at stalled replication forks, promoting fork reversal and template switching. It interacts with PCNA, RAD51, and PARP1, and its HIRAN domain recognizes ssDNA to facilitate fork remodeling. HLTF regulates PCNA ubiquitination and RAD51 recruitment, working in concert with SHPRH to enable lesion bypass. This positions HLTF upstream of key effectors such as CHK1, CHK2, ??H2AX, 53BP1, BRCA1, and BRCA2, integrating chromatin remodeling with homologous recombination and checkpoint signaling.
In HEK293T cells, HLTF disruption offers a versatile model to probe the chromatin remodeling contributions to genome stability. The cell line??s permissiveness for replication stress experiments, combined with the HLTF loss-of-function, enhances sensitivity to genotoxic agents, facilitating dissection of fork protection mechanisms. The system??s high transfectability further supports complementation studies, enabling detailed structure?Cfunction analyses of HLTF domains and its interactions with partner proteins.
Common applications include western blotting and RT-qPCR for knockout validation, ??H2AX immunofluorescence to assess double-strand break accumulation, comet assays for bulk DNA damage, and DNA fiber labeling to monitor replication fork dynamics. Clonogenic survival and apoptosis assays are used to evaluate chemosensitivity, while flow cytometry and RNA-seq reveal cell cycle and transcriptomic changes. ChIP-qPCR can interrogate chromatin association. This product is well-suited for studies of DNA damage tolerance, replication stress, and tumor suppression, as well as drug discovery efforts targeting the DNA damage response. For technical inquiries, contact Ascent Research.
