The E8l enhancer-Cd8a promoter-Kozak-Cre-rBG pA cassette was cloned into intron 1 of ROSA26 in reverse orientation.

The CD8A gene encodes the CD8α chain of the CD8 T-cell coreceptor, a cell-surface glycoprotein essential for cytotoxic T lymphocytes (CTLs), or CD8⁺ T cells, which mediate MHC class I-restricted antigen recognition to eliminate infected or cancerous cells. The CD8 coreceptor is typically a heterodimer of CD8α and CD8β chains (encoded by the CD8A and CD8B genes, respectively) on CD8⁺ T cells, but CD8α-α homodimers are also expressed, notably on subsets of intestinal intraepithelial lymphocytes (IELs) and natural killer (NK) cells. The E8I enhancer is a critical cis-regulatory element located within the CD8 locus that is crucial for the gene expression of CD8A and, to a lesser extent, CD8B. E8I is active in mature CD8 single-positive thymocytes, CD8⁺ T cells, and CD8α-α⁺ IELs in the gut. Its primary function is the maintenance of high-level CD8α expression upon CD8⁺ T-cell activation, a process dependent on transcription factors like Runx3, and it is also required for the upregulation of the CD8α-α homodimer associated with memory T-cell differentiation and CD4 CTL generation. The H11-E8I-Cd8a-iCre(BALB/c) mouse is generated by integrating an iCre recombinase (codon-optimized Cre) expression cassette, driven by the E8I enhancer and Cd8a promoter, into the H11 safe harbor locus via gene editing. When crossed with loxP-flanked mice, the offspring are expected to exhibit Cre-mediated recombination in CD8+ T cells, the key cytotoxic effectors of the immune system.

Emcn is located on chromosome 3 of mice. SgRNA and ssDNA were designed using Nuclease Technology; Emcn conditional knockout mice were obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Cd8a is located on chromosome 6 of mice. Nuclease Technology was used to design sgRNA; Cd8a knockout mice were obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Cd8a is located on chromosome 6 of mice. Nuclease Technology was used to design sgRNA; Cd8a knockout mice were obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Cd8a is located on chromosome 6 of mice. SgRNA and ssDNA were designed using Nuclease Technology; Cd8a conditional knockout mice were obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Ccdc70 is located on chromosome 8 of mice. Nuclease Technology will be used to design sgRNA; Ccdc70 knockout mice will be obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.