Dhfr Knockout RAW 264.7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population with disruption of the Dhfr gene in the RAW 264.7 murine macrophage cell line. This loss-of-function model enables investigation of dihydrofolate reductase (DHFR) within an innate immune effector cell context. As a polyclonal pool, the population reflects a range of editing outcomes suitable for pooled functional assays, providing a versatile tool for studying folate metabolism, nucleotide biosynthesis, and antifolate drug sensitivity.
The host RAW 264.7 cell line, derived from BALB/c mice, is a widely used monocyte/macrophage-like model exhibiting phagocytic activity, cytokine production, and antigen presentation. These cells are commonly employed to study innate immune signaling, inflammatory responses, and macrophage-driven pathologies. Their robust proliferation and tractability for genetic manipulation make them an established system for investigating metabolic regulation in immune cells.
DHFR catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, a critical one-carbon carrier for thymidylate and purine biosynthesis. DHFR expression is regulated upstream by transcription factors E2F, Sp1, and c-Myc, while its activity is inhibited by methotrexate and influenced by folate availability. Downstream, DHFR sustains nucleotide pools and supports thymidylate synthase and purine biosynthetic enzymes, interacting with NADPH, methotrexate, and SHMT1. Within the folate cycle, DHFR collaborates with MTHFR, MTHFD1, and serine hydroxymethyltransferase to maintain one-carbon flux and cell cycle progression.
Disruption of Dhfr in RAW 264.7 macrophages abrogates de novo nucleotide synthesis, causing reduced proliferation and heightened sensitivity to antifolates like methotrexate. This model recapitulates metabolic vulnerabilities of DHFR-deficient cells and can be leveraged to probe the biosynthetic demands of macrophage functions, including phagocytosis and cytokine secretion, which rely on adequate nucleotide pools. Consequently, it offers insights into how one-carbon metabolism supports innate immune cell activity.
Applications include mechanistic investigations of methotrexate resistance, screening of novel antifolate agents, and dissection of folate-dependent cell cycle control. Typical assays involve MTT proliferation, methotrexate cytotoxicity, western blotting for DHFR, RT-qPCR for pathway gene expression, cell cycle flow cytometry, and nucleotide pool quantification. For technical inquiries and ordering, please contact Ascent Research.
