Description

The Mlh1 Knockout CT26.WT Cell Line is a CRISPR/Cas9-edited knockout cell line engineered for loss-of-function studies of the Mlh1 gene in a murine colorectal cancer background. Derived from the CT26.WT host cell line, this product provides a stable, target-gene-disrupted population optimized for investigating DNA mismatch repair deficiency and its downstream consequences. The knockout model enables researchers to dissect the molecular mechanisms underlying microsatellite instability and cancer predisposition without the confounding effects of transient silencing or pharmacological inhibition. By introducing defined genetic disruption via CRISPR/Cas9, the cell line offers a renewable and consistent experimental system for long-term studies.

The CT26.WT cell line originates from a chemically induced colorectal adenocarcinoma in BALB/c mice, serving as a well-characterized syngeneic model for colorectal carcinoma and tumor immunology. These cells are widely utilized in preclinical research due to their reproducibility in forming tumors in immunocompetent BALB/c hosts, facilitating translational studies of the tumor microenvironment and immunotherapeutic responses. The Mlh1 knockout derivative retains the parental line’s core phenotypic traits while incorporating targeted disruption of a critical DNA repair gene, creating a platform to interrogate genotype?Cphenotype correlations in a host-relevant setting.

MLH1 functions as a central component of the MutL?? heterodimer alongside PMS2, recognizing and directing repair of replication-dependent base mismatches and insertion?Cdeletion loops. Mlh1 disruption abolishes functional mismatch repair, leading to the accumulation of spontaneous mutations and microsatellite instability. This protein complex interacts with MSH2?CMSH6 (MutS??) and EXO1, and is loaded onto DNA at replication foci by PCNA, coupling repair to the replication machinery. Upstream, MLH1 expression is regulated by TP53 and MYC transcriptional programs and is susceptible to epigenetic silencing through promoter hypermethylation. Loss of MLH1 impairs apoptosis signaling in response to DNA damage, allowing survival of genomically unstable cells. Thus, the knockout systematically dismantles the multi-component repair network, from lesion recognition by MutS?? to excision and re-synthesis steps.

In the CT26.WT context, ablation of Mlh1 converts an otherwise microsatellite-stable (MSS) tumor line into a microsatellite instability-high (MSI-H) model, mirroring key features of Lynch syndrome and a subset of sporadic colorectal cancers. The elevated mutation burden generates neoantigens that can be recognized by the adaptive immune system, making this cell line particularly valuable for syngeneic immunotherapy studies. When implanted into BALB/c mice, Mlh1-knockout CT26.WT tumors exhibit altered growth kinetics, enhanced immunogenicity, and differential responses to checkpoint blockade compared to the wild-type counterpart. This model thus bridges DNA repair biology and immuno-oncology, facilitating exploration of how genomic instability shapes tumor evolution and therapeutic vulnerability.

This knockout cell line supports a broad array of research applications, including mechanistic studies of mismatch repair, microsatellite instability profiling, and drug sensitivity screening using DNA-damaging agents such as SN38 or temozolomide. It is compatible with western blotting to confirm MLH1 loss, PCR-based MSI fragment analysis, whole-exome sequencing for mutation signature deconvolution, and viability or apoptosis assays following genotoxic stress. In vivo, the line enables syngeneic tumor growth assays coupled with immune profiling by flow cytometry or transcriptomic analysis via RNA-seq to dissect tumor?Cimmune interactions. Researchers studying hereditary nonpolyposis colorectal cancer (HNPCC) mechanisms, MSI-high immunobiology, or developing novel therapeutics for mismatch repair-deficient tumors will find this model a robust tool. For further technical details and custom inquiries, contact Ascent Research.