Abstract: 【Abstract】 Objective To investigate the abnormal physiological phenotype of hair follicles in mice with the Klhl24 gene initiation codon mutation, and to provide a basis for elucidating the regulatory mechanism of hair follicle development by the gene. Methods A Klhl24c.3G/T male mouse carrying a heterozygous mutation Klhl24c.3G>T in the initiation codon of the Klhl24 gene was produced by using clustered regularly interspaced short palindromic repeats （CRISPR）/Cas9 technology, and mated with 2 wild-type female mice. Then, the littermate mice were genotyped. The Klhl24c.3G/T male mice and wild-type male mice served as the experimental group and control group respectively, and there were more than 3 mice in each group. On days 21 （the first telogen phase of hair cycle） and 45 （the second telogen phase of hair cycle） after birth, the tape sticking experiment was carried out. Immunohistochemical study of Ki67 expression was performed in skin tissues from the back of the mice, scanning electron microscopy and transmission electron microscopy were conducted to observe the hair root at the bottom of the hair shaft and the structure of mitochondria in skin tissues respectively, and a terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling （TUNEL） kit was used to detect apoptosis in hair follicles. The Dunnett-t test was used for comparisons between the experimental group and control group. Results The number of shed hair shafts per 0.25 cm2 of the tape at the first and second telogen phases was significantly higher in the Klhl24c.3G/T mice （1 224 ± 51.08, 1 514 ± 72.15 respectively） than in the wild-type mice （320 ± 55.68, 125 ± 2.86, t = 11.96, 19.24, respectively, both P < 0.001）. Scanning electron microscopy showed clubbed hair roots at the bottom of the shed hair shaft at the first telogen phase. On day 59 after birth, there was no new hair shaft growing on the back of the wild-type mice and the hair follicles were still at the telogen phase, while new hair shafts had grown on the back of the Klhl24c.3G/T mice and the hair follicles had entered the next anagen phase. At the first telogen phase （i.e., on day 21）, transmission electron microscopy showed disordered structures of mitochondrial cristae in cells of the hair follicles in the skin tissues from the back of the Klhl24c.3G/T mice, and the number of apoptotic cells in hair follicles （12 ± 1.15） was significantly higher in the Klhl24c.3G/T mice than in the wild-type mice （3 ± 0.63, n = 8, t = 6.874, P < 0.001）. Conclusion The abnormal hair phenotype of the Klhl24c.3G/T mice mainly manifested as decreased anchoring ability of hair shafts at the telogen phase, precocious entry into the anagen phase, abnormal structure of mitochondria in hair follicle cells, and increased number of apoptotic cells.

Key words: Hair follicle, Mice, mutant strains, Alopecia, Mitochondria, Apoptosis, Klhl24 gene