〔Abstract〕 Objective To explore the construction and identification of miR-100 gene knockout mouse model. Methods The introduced miR-100flox/floxmice were mated with EIIa-Cre mice to produce F1 heterozygous mice. The male and female mice with miR-100flox/-genotype were further mated to obtain F2 generation mice. When the mice were 1 week old, the tail tissueswere cut to extract DNA,the target gene fragment was amplified by PCR,and the genotype was determined by agarose gel electrophoresis. Results Among them, there were miR-100 knockout homozygous mice. If the obtained knockout homozygous mice were mated, more knockout homozygous mice could be obtained. By cutting the mouse ears to extract the RNA,the mouse RNA could be extracted without affecting the life of the mouse. Therefore, the ears of miR-100 knockout homozygous mice, miR-100flox/floxmice, and miR-100 knockout heterozygous mice were cut to extract RNA,and then qPCR was performed to verify the expression of miR-100. The breeding and identification of miR-100 gene knockout mice were successful, and stable genetic knockout mice were obtained. Three genotypes were found in F2 generation mice mated with F1 heterozygous mice: miR-100flox/flox,miR-100flox/-,and miR-100-/-. The genotypes of the offspring mice were successfully identified by PCR. qPCR results showed that the expression of miR-100 in the ears of the whole-body knockout homozygous mice was almost zero. Conclusion In this study, a mouse model of whole-body deletion of miR-100 gene is successfully cloned, providing a basis for subsequent experiments.