Description

The Rnf31 Knockout MC-38 Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the MC-38 murine colon adenocarcinoma cell line, designed to disrupt the Rnf31 gene. This gene encodes HOIP (HOIL-1-interacting protein), the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), a master regulator of linear ubiquitination and NF-??B signaling. By introducing targeted gene disruption via CRISPR/Cas9, this cell line provides a stable loss-of-function model for dissecting the roles of linear ubiquitination in inflammatory signaling, cell death, and tumor immunity.

The parental MC-38 cell line is an epithelial tumor cell line originally established from a chemically induced colorectal carcinoma in C57BL/6 mice. As a syngeneic colon adenocarcinoma model, MC-38 is extensively utilized in cancer immunology and preclinical colorectal cancer studies. It retains key tumor cell characteristics, including oncogenic signaling and immune-modulatory responsiveness, offering a well-characterized background for gene-edited models focused on tumor-intrinsic pathways.

Rnf31 encodes HOIP, which partners with SHARPIN and HOIL-1L (RBCK1) to form LUBAC, the sole E3 ligase generating Met1-linked linear ubiquitin chains. LUBAC is recruited to TNFR1, IL-1R, and TLR complexes, where it linearly ubiquitinates NEMO and RIPK1. This ubiquitination activates the IKK complex, driving NF-??B nuclear translocation and transcription of pro-inflammatory cytokines (e.g., TNF-??, IL-6, IL-1??). Moreover, LUBAC-mediated linear ubiquitination restrains TNF-??-induced apoptosis and necroptosis. Rnf31 disruption abolishes these functions, abolishing linear ubiquitination, impairing NF-??B responses, and sensitizing cells to cell death.

In MC-38 colon adenocarcinoma cells, Rnf31 knockout severely disrupts tumor cell-intrinsic inflammatory signaling and cell death regulation. This model allows dissection of how linear ubiquitination balances NF-??B survival signals and programmed cell death in colorectal cancer. It is ideal for studying immunogenic cell death, tumor-immune interactions, and LUBAC??s role in inflammatory bowel disease-related carcinogenesis. The knockout cell line also facilitates screening of LUBAC inhibitors targeting the catalytic subunit.

Researchers can employ western blotting for linear ubiquitin chains and phospho-IKK, RT-qPCR for NF-??B targets (Il6, Tnf), immunofluorescence for p65 nuclear translocation, and flow cytometry (Annexin V/PI) to assess cell death. Co-immunoprecipitation of RIPK1 with NEMO, TNF-?? sensitivity assays, and cytokine ELISA further support mechanistic studies. Applications span tumor immunity, inflammatory bowel disease, and LUBAC-targeted drug discovery. For further information, contact Ascent Research.