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

• Published in: Journal of cellular biochemistry 2019-07, Vol.120 (7), p.11033-11043
• Main Authors: Nasseri, Sherko, Nikkho, Bahram, Parsa, Sara, Ebadifar, Asghar, Soleimani, Farzad, Rahimi, Karim, Vahabzadeh, Zakaria, Khadem‐Erfan, Mohammad Bagher, Rostamzadeh, Jalal, Baban, Babak, Banafshi, Omid, Assadollahi, Vahideh, Mirzaie, Sako, Fathi, Fardin
• Format: Article
• Language: English
• Subjects: amelogenesis imperfecta; Autosomal recessive inheritance; Body size; Calcification; Casein; Casein kinase I; Cervix; CRISPR; CRISPR‐Cas system; Cytoskeleton; Dental enamel; Dismantling; Enamel; family with sequence similarity 83 member H protein; Filaments; Heredity; Inbreeding; Keratin; Kinases; knockout mice; Life span; Mandible; Mice; Molars; Morphology; Nonsense mutation; outbred mice; Phenotypes; Polymerase chain reaction; Rodents; Signs and symptoms; Teeth
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.28381

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. Homozygous FAM83H knockout (KO) outbred mice compared with wild‐type mice had the smaller size, scruffy cover, generally decreased activity, and delayed eye‐opening but showed normal lifespan and reproductive ability. KO of FAM83H had controversial effects on the development of teeth and the formation of enamel. The phenotype defect in dental development and enamel formation were seen in three mice among four generations.