Generation of Fam83h knockout mice by CRISPR/Cas9‐mediated gene engineering

Abstract

Family with sequence similarity 83 member H (FAM83H) protein-coding geneplay an essential role in the structural organization, calcification of developing enamel, and keratin cytoskeleton disassembly by recruiting Casein kinase 1 alpha (CSNK1A1) to keratin filaments. In this study, we have applied CRISPR Cas9 nickase (D10A) to knockout (KO) the Fam83h gene in NMRI outbred mice. We generated homozygous Fam83h KO mice (Fam83h Ko/Ko ) through a premature termination codon, which was validated by Sanger sequencing in F0 generation. Next, we also bred the FAM83H KO for two generations. Reverse-transcription polymerase chain reaction and Western blot analysis approved the Fam83h KO mice. The Fam83h KO mice had evidence of normal morphology at the cervical loops, secretory and maturation stages, and mandibular molars. In comparison with the normal wild-type mice (Fam83h W/W ), the F2 homozygous KO (Fam83h Ko/Ko ) had sparse, scruffy coats with small body size and decreased general activity. Also, they had the natural reproductive ability and natural lifespan. In addition, delay in opening the eyes and dry eyes among infant mice were seen. The F1 heterozygous mice looked comparable to the normal wild-type mice (Fam83h W/W ), which showed autosomal recessive inheritance of these phenotypes. The KO of FAM83H had controversial effects on the development of teeth and the formation of enamel.The phenotype defect in dental development and the enamel formation were seen in three mice among four generations. It can be concluded that null FAM83H in outbred mice not only showed the reported phenotypes in null inbred mouse but also showed normal lifespan and reproductive ability; dental deficiency in three homozygous mice; and the symptoms that were similar to the symptoms of dry eye syndrome and curly coat dog syndrome in all four evaluated KO generations. K E Y W O R D Samelogenesis imperfecta, CRISPR-Cas system, family with sequence similarity 83 member H protein, knockout mice, outbred mice

FAM83H and SCRIB stabilize β-catenin and stimulate progression of gastric carcinoma

“…In particular, knock-out of FAM83H retarded overall growth of mice and induced malformation of digits, AGING and abnormal teeth development was not part of the phenotype of all the knock-out mice [3]. These aberrant phenotypes in FAM83H knock-out mice suggest that FAM83H can have a pleiotropic role in cell proliferation and the molecules interacting with FAM83H might be involved in various roles of FAM83H [3]. In this aspect, the oncogene MYC is presented as a transcriptional regulator of FAM83H in hepatocellular carcinomas [4].…”

“…However, the effects of knocking out FAM83H in mice are not restricted to teeth development [3]. In particular, knock-out of FAM83H retarded overall growth of mice and induced malformation of digits, AGING and abnormal teeth development was not part of the phenotype of all the knock-out mice [3].…”

“…However, the effects of knocking out FAM83H in mice are not restricted to teeth development [3]. In particular, knock-out of FAM83H retarded overall growth of mice and induced malformation of digits, AGING and abnormal teeth development was not part of the phenotype of all the knock-out mice [3]. These aberrant phenotypes in FAM83H knock-out mice suggest that FAM83H can have a pleiotropic role in cell proliferation and the molecules interacting with FAM83H might be involved in various roles of FAM83H [3].…”

FAM83H mutation analysis and its expression in rat tooth germs and human dental cells

“…As the truncated FAM83H protein causes ADHCAI and the mutational study of FAM83H revealed the importance of the C-terminal portion for tooth enamel calci cation [22], we propose that the C-terminus of FAM83H is the core function region. FAM83H null mice and transgenic mice overexpression FAM83H did not show phenotype in enamel and dentin development, suggesting that the mechanism of FAM83H mutant ADHCAI was gain-of -function or dominant negative effect [23][24][25]. Recently, Wang et al generated a mouse model expressing a truncated FAM83H protein that recapitulated the ADHCAI-causing human FAM83H p.Tyr297* mutation, which displayed obvious enamel malformations.…”

Characterisation of the biochemical and cellular roles of native and pathogenic amelogenesis imperfecta mutants of FAM83H

“…FAM83H is not expected to be secreted into the enamel matrix as it lacks a secretory signal peptide and is therefore expected to have intracellular roles in ameloblasts. However, whether FAM83H mainly functions during the pre-secretory, secretory or maturation stage of amelogenesis remains unclear [ 23 , 33 ].…”