The Dcaf15 Knockout RAW 264.7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the RAW 264.7 mouse macrophage line, offering targeted disruption of the Dcaf15 gene. This polyclonal model enables robust loss-of-function analysis of DCAF15, a substrate receptor for the CUL4-DDB1 (CRL4) E3 ubiquitin ligase that directs proteasomal degradation of key substrates such as RBM39. The population approach avoids clonal artifacts, providing a heterogeneous system ideal for studying ubiquitin-proteasome dynamics in a macrophage background.
The parental RAW 264.7 line is an Abelson murine leukemia virus-transformed macrophage model extensively used in immunological research. These cells exhibit characteristic phagocytic activity, inflammatory signaling, and antigen presentation capabilities, making them a versatile platform for investigating innate immunity. The line??s genetic tractability and well-defined signaling networks facilitate the integration of CRISPR-mediated knockouts with functional macrophage assays, ensuring physiologically relevant readouts.
DCAF15 functions as a substrate-recognition subunit within the CRL4 complex, which assembles with CUL4A or CUL4B, DDB1, and the RING protein RBX1. Complex activation depends on cullin neddylation through the NEDD8 conjugation pathway and is modulated by the COP9 signalosome. DCAF15 specifically recruits substrates like RBM39 for polyubiquitination and subsequent degradation by the 26S proteasome, a process responsive to DNA damage signals. Disruption of Dcaf15 stabilizes RBM39 and other proteasomal targets, leading to altered RNA splicing, cell cycle progression, and apoptosis. Downstream effects are mediated by accumulated RNA splicing factors and impaired turnover of cell cycle regulators.
In the macrophage context, loss of Dcaf15 is predicted to impair CRL4DCAF15-mediated proteostasis, with potential consequences for phagocytosis, cytokine secretion, and antigen presentation. Stabilized RBM39 may reprogram alternative splicing of genes involved in immune responses, while cell cycle and apoptosis disturbances could influence macrophage proliferation and survival under inflammatory conditions. This model is thus valuable for dissecting the role of the ubiquitin-proteasome system in innate immunity and for modeling CRL4 dysfunction in myeloid cells, relevant to cancer biology and hematological malignancies. Representative pathway components include CUL4A/B, DDB1, DCAF15, RBX1, E2 ubiquitin-conjugating enzymes, the 26S proteasome, and RBM39.
Researchers can employ these knockout cells in diverse experimental workflows. Typical applications include analyzing ubiquitin-proteasome system function via western blotting, co-immunoprecipitation, and ubiquitination assays; studying RBM39-dependent splicing with RNA-seq and RT-qPCR; and measuring macrophage activity through phagocytosis and cytokine secretion assays. Flow cytometry can assess surface markers and apoptosis. Additionally, the cells are suited for screening sulfonamide drugs that target DCAF15, enabling investigation of mechanism of action and resistance. For technical inquiries, please contact Ascent Research.
