中国生物工程杂志

Objective: Programmed death ligand-1 (PD-L1) is an important factor in the immunoregulatory pathway and is one of the important targets in anti-tumor immunotherapy. The PD-L1 knockout mouse model was successfully constructed using CRISPR/Cas9 technology, and its phenotype was initially analyzed.Methods: Cas9 and sgRNA vectors were constructed and transcribed to obtain RNA. RNA was injected into C57BL/6 mouse fertilized eggs by microinjection, and F₀ generation positive mice were obtained. F₀ generation mice were mated with wild type C57BL/6 mice to obtain F₁ generation heterozygous mice, and F₂ generation homozygous mouse strains were obtained by self-crossing of F₁ generation mice. Subsequently, the expression of PD-L1 gene at the mRNA and protein levels was detected by Real-Time PCR and flow cytometry, respectively.Results: Real-Time PCR and flow cytometry showed that the PD-L1 mRNA expression level and protein expression on PD-L1 homozygous mice with only background signals were significantly decreased, compared with wild-type C57 mice. It was confirmed that the PD-L1 knockout mouse strain was successfully constructed, which provided a new mouse model for PD-L1 gene function research in vivo.