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.

The TGA stop codon of the mouse Cd7 gene is replaced with Furin-GSG-T2A-Dre. The Furin sequence helps to remove the 2A residues from the upstream protein, while the GSG linkers can provide the necessary space to ensure proper protein folding and self-cleavage of the 2A sequence.

For the Kl model, the coding sequence of the exon 1 plus partial intron 1 was replaced with the Dre-rBG pA cassette. This model expresses Dre recombinase driven by the regulatory elements of the mouse Cd34 gene.

The CRISPR/Cas system has revolutionized genome editing by enabling precise and efficient genetic modifications in vivo ^[1-2]. This system consists of two principal components: the CRISPR-associated (Cas) endonucleases, which introduces site-specific double-strand breaks in DNA, and a guide RNA (gRNA), a short RNA sequence embedded within a scaffold that directs Cas to its genomic target via sequence complementarity ^[3]. H11-CAG-hCas mice carry a human codon-optimized Streptococcus pyogenes CRISPR-associated protein 9 (SpCas9) expression cassette, driven by the ubiquitous CAG promoter. This configuration ensures systemic expression of Cas protein across tissues. Notably, constitutive Cas expression in these mice does not elicit detectable toxicity, morphological abnormalities, tumorigenesis, or activation of DNA damage or apoptotic pathways. In the presence of sequence-specific single-guide RNAs (sgRNAs), this model enables efficient and simultaneous editing of single or multiple genes. H11-CAG-hCas mice are viable, fertile, and well-suited for a wide range of genome engineering applications.

The TGA stop codon was replaced with the "P2A-Dre" cassette. Dre recombinase is expressed under the regulatory control of Itgax gene elements.

The TGA stop codon of the mouse Kit gene is replaced with loxP-6*SV40 pA-loxP-T2A-DTR-P2A-Dre. When Kit-LSL-T2A-DTR-P2A-Dre mice are crossed with Cre mice, the 6*SV40 pA sequence in the offspring mice is deleted, and DTR (diphtheria toxin receptor) and Dre are expressed.

The DTR system is a novel cell lineage ablation system based on the expression of the diphtheria toxin receptor (DTR) in transgenic mice and the administration of diphtheria toxin (DT). By knocking in the DTR gene at the target gene locus (usually a marker gene), selective ablation of cells expressing specific genes can be achieved ^[1]. When the target gene begins to be expressed in cells of mice that have been transgenically modified to express DTR, injection of DT can induce the death of these cells, resulting in apoptosis. The core of the DTR system is the toxic effect of DT, an exotoxin that, once it enters cells via its receptor, can catalyze the transfer of the ADP-ribosyl moiety from NAD+ to elongation factor 2 (EF-2) due to the ADP-ribosyltransferase activity present in its C-domain, resulting in EF-2 inactivation and inhibition of protein synthesis, ultimately leading to cell death ^[2]. Due to species differences in the sequence of the DTR gene, the diphtheria toxin receptor in mice cannot bind to DT ^[3]. This can be overcome by knocking in the primate DTR gene into the mouse genome, allowing specific cells to express DTR and enabling DT to enter specific cells upon administration, and resulting in cell-specific ablation. RC-LR-DTR mice are generated by combining the DTR system with the Cre-loxP and Dre-rox conditional recombinase systems. When these mice are crossed with Cre and Dre mice, DTR can be expressed specifically in tissues to induce tissue- and cell-specific ablation. Heterozygous RC-LR-DTR mice are viable and fertile, and homozygous RC-LR-DTR mice are also viable.

The "P2A-Flpo" cassette was inserted upstream of TAA stop codon. Flpo recombinase is expressed under the regulatory control of Trhr gene elements.