The P2A-3xSV40 NLS-mScarlet cassette was inserted upstream of TGA stop codon. Nuclear-localized mScarlet is expressed under the regulatory control of Alpl gene elements in this mouse model. This localization is achieved through the Nuclear Localization Signal (NLS), which efficiently targets mScarlet to the cell nucleus. This enables mScarlet fluorescence protein nuclear tracing studies.

The TAA stop codon was replaced with the P2A-EGFP-T2A-iCre cassette. Cre recombinase and enhanced green fluorescent protein (EGFP) are expressed under the regulatory control of Alas2 gene elements.

The ADGRL2 gene (Adhesion G Protein-Coupled Receptor L2), also known as latrophilin 2, encodes a member of the adhesion G protein-coupled receptor (aGPCR) family, which are characterized by a long N-terminal domain involved in cell-cell and cell-matrix interactions ^[1]. The encoded protein, ADGRL2, is involved in various physiological processes, including cell adhesion, neuronal development, regulation of exocytosis (e.g., as a low-affinity receptor for alpha-latrotoxin), and maintaining intestinal homeostasis ^[2]. It is expressed in numerous tissues, with notable expression in the central nervous system (neurons, hippocampus), intestinal epithelium, and specifically, its expression is strongly upregulated during keratinocyte differentiation in epidermal tissue ^[3]. Dysregulation or variations in ADGRL2 have been associated with a range of conditions, including neurodegenerative diseases (like Alzheimer's and Parkinson's), inflammatory bowel diseases (Crohn's disease, ulcerative colitis), certain autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis), and even metabolic syndrome and cocaine use disorder. Adgrl2-3xGGGGS-mCherry mice are constructed by replacing the partial exon 1 coding region of the mouse Adgrl2 gene with HA signal peptide - HA tag - Mouse Adgrl2 CDS (without signal peptide) - 3xGGGGS - mCherry - rBG pA cassette using gene editing technology. The Adgrl2-3xGGGGS-mCherry mouse carries a red fluorescent protein (mCherry) expression cassette, making it a precise research model that maintains protein function while offering fluorescence visualization. This model is valuable for several key areas of study. For instance, it can be used for the spatio-temporal dynamic analysis of the Adgrl2 gene expression profile. Researchers can also utilize it for investigating neuronal development and synapse formation mechanisms. Furthermore, it enables live-animal dynamic tracking and real-time imaging observation, providing invaluable insights. Lastly, this model is well-suited for systematic studies of protein interaction networks and downstream signaling pathways.

The AGRP gene encodes Agouti-related protein (AgRP), a neuropeptide synthesized by AgRP/NPY neurons predominantly located in the arcuate nucleus of the hypothalamus, as well as in the kidneys and adrenal glands. The expression of AGRP is modulated by various factors, including nutritional status and hormonal signals. Notably, AGRP expression is markedly upregulated during periods of starvation and rapidly downregulated following refeeding. AgRP is exclusively synthesized in the ventromedial part of the arcuate nucleus within neuropeptide Y (NPY)-containing cells, where it is co-expressed with NPY. This neuropeptide plays a pivotal role in enhancing appetite, reducing metabolic rate, and decreasing energy expenditure, making it one of the most potent and enduring appetite stimulators. AgRP exerts its orexigenic effects by antagonizing melanocortin receptor 4 (MC4R), thereby promoting food intake and inhibiting energy expenditure, which is crucial for weight regulation. Mutations in the AGRP gene have been implicated in conditions such as late-onset obesity and anorexia nervosa, underscoring its significant role in energy homeostasis and body weight control. The Agrp-IRES-CreERT2-P2A-tdTomato mouse model was generated by integrating the IRES-CreERT2-P2A-tdTomato gene expression cassette into the endogenous Agrp locus via gene editing technology. Under the control of the mouse endogenous Agrp gene regulatory elements, this mouse expresses tamoxifen-inducible CreERT2 recombinase. Additionally, the cassette includes a red fluorescent protein (tdTomato) for lineage tracing of Agrp-positive cells. In the absence of tamoxifen, CreERT2 recombinase remains cytoplasmic. Upon tamoxifen administration, CreERT2 translocates to the nucleus to mediate recombination. When Agrp-IRES-CreERT2-P2A-tdTomato mice are crossed with mice containing loxP sites, tamoxifen induction can trigger Cre recombinase-mediated sequence recombination between loxP sites in AgRP-positive neurons of the offspring.

The TAG stop codon was replaced with "3xGGGGS-EGFP" cassette. Enhanced green fluorescent protein (EGFP) is expressed under the regulatory control of Adgrg3 gene elements. This model enables EGFP fluorescence protein tracing studies.

The Cdkn2a (cyclin-dependent kinase inhibitor 2A) gene encodes proteins including p16^INK4a and plays an important role in cell cycle regulation and cellular senescence. p16^INK4a is one of the commonly used markers of cellular senescence. It is expressed in various tissues and cell types and can be upregulated during senescence, injury, or cellular stress ^[1-3]. Luciferase is an enzyme that produces a bioluminescent signal in the presence of an appropriate substrate. In this model, the endogenous stop codon of the mouse Cdkn2a gene was replaced with a P2A-Luciferase-T2A-iCre expression cassette using gene-editing technology. Luciferase and iCre are expected to be expressed in senescence-associated cells, tumor cells, and other Cdkn2a-positive cells. When Cdkn2a-P2A-Luciferase-T2A-iCre mice are crossed with mice carrying loxP sites, the offspring can undergo iCre-mediated recombination between loxP sites in Cdkn2a-positive cells.

The Cdkn2a (cyclin-dependent kinase inhibitor 2A) gene encodes proteins including p16^INK4a and plays an important role in cell cycle regulation and cellular senescence. p16^INK4a is one of the commonly used markers of cellular senescence. It is expressed in various tissues and cell types and can be upregulated during senescence, injury, or cellular stress ^[1-3]. In this model, the endogenous stop codon of the mouse Cdkn2a gene was replaced with the loxP-AA-6×SV40 pA-loxP-TG-P2A-DTR-T2A-tdTomato expression cassette using gene-editing technology. The p16 transcript can theoretically continue translation into the loxP sequence, resulting in an additional amino acid sequence at the C-terminus, followed by a new termination signal formed by the inserted “AA” sequence. Following Cre-mediated excision of the LSL element, the endogenous Cdkn2a regulatory elements drive the expression of DTR (diphtheria toxin receptor) and tdTomato fluorescent protein. Cdkn2a-positive cells express tdTomato, and subsequent administration of diphtheria toxin (DT) selectively depletes DTR-expressing cells. DTR and tdTomato are expected to be expressed in senescence-associated cells, tumor cells, and other Cdkn2a-positive cells.

The TAA stop codon was replaced with the P2A-tdTomato-IRES-loxP-6xSV40 pA loxP-DTR cassette. Upstream of the DTR (diphtheria toxin receptor) gene lies a loxP-Stop-loxP cassette that blocks DTR transcription and expression. In the absence of Cre recombinase, the Ctnnd2 promoter drives tdTomato expression, labeling Ctnnd2-positive cells with red fluorescence. 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 TAG stop codon was replaced with P2A-EGFP-IRES-loxP-6xSV40 pA-loxP-DTR cassette. Upstream of the DTR (diphtheria toxin receptor) gene lies a loxP-Stop-loxP cassette that blocks DTR transcription and expression. In the absence of Cre recombinase, the Ccr7 promoter drives EGFP expression, labeling Ccr7-positive cells with green fluorescence. 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 IRES-CreERT2-P2A-EGFP-WPRE-BGH pA cassette was inserted downstream of the TGA stop codon. CreERT2 recombinase and enhanced green fluorescent protein (EGFP) are expressed under the regulatory control of Car1 gene elements. This model is a Tamoxifen-inducible Cre mouse, and when crossed with mice containing loxP sites, the offspring mice are expected to undergo sequence recombination between loxP sites mediated by Cre recombinase in Car1-positive cells following Tamoxifen induction. In addition to studies of Cre recombinase-mediated gene recombination, this model can also be used for EGFP fluorescent protein tracing studies.