The TGA stop codon was replaced with the P2A-iCre-WPRE-BGH pA cassette. Cre recombinase is expressed under the regulatory control of Cd68 gene elements.

The CD68 gene encodes a transmembrane glycoprotein primarily expressed on the surface of macrophages and other mononuclear phagocytes, with high expression levels in monocytes and tissue macrophages. The CD68 protein belongs to the lysosome-associated membrane protein (LAMP) family, localizes mainly to lysosomes and endosomes, and participates in phagocytosis, antigen presentation, and inflammatory responses. As a commonly used immunohistochemical marker, CD68 is widely employed to identify macrophages in the spleen, liver, and inflammatory or tumor microenvironments. Dysregulation of its expression is associated with chronic inflammatory diseases, atherosclerosis, and cancer, with tumor-associated macrophages (TAMs) playing a key role in disease progression. The Cd68-P2A-CreERT2 mouse model was constructed using gene editing technology to integrate the tamoxifen-inducible CreERT2 recombinase expression element into the endogenous mouse Cd68 gene. Without tamoxifen treatment, the CreERT2 recombinase remains in the cytoplasm; only upon tamoxifen induction can it translocate to the nucleus and initiate recombination. When Cd68-P2A-CreERT2 mice are crossed with mice containing loxP sites, tamoxifen induction is expected to trigger Cre-mediated recombination between loxP sites in monocytes and macrophages of the offspring.

The loxP-3*SV40 pA-loxP-IRES-DTR-rBG pA cassette was inserted downstream of the TGA stop codon. Upstream of the DTR (diphtheria toxin receptor) gene lies a loxP-Stop-loxP cassette that blocks DTR transcription and expression. Upon Cre recombination, Cre-mediated deletion of the loxP-flanked stop (LSL) element enables DTR expression in Cre-positive cells. Intravenous administration of diphtheria toxin (DT) then selectively ablates these cells.

Midn is located on chromosome 10 of mice. SgRNA and ssDNA were designed using Nuclease Technology; Midn conditional knockout mice were obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Cd68 is located on chromosome 11 of mice. SgRNA and ssDNA were designed using Nuclease Technology; Cd68 conditional knockout mice were obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Sumf2 is located on chromosome 5 of mice. Nuclease Technology will be used to design sgRNA; Sumf2 knockout mice will be obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Cd68 is located on chromosome 11 of mice. Nuclease Technology will be used to design sgRNA; Cd68 knockout mice will be obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.