Aging is a physiological change that leads to a decline in biological functions from metabolic stress. To investigate the effect of aging on mandibular bone formation, we created SAMP1/Klotho-deficient mice and performed micro-computed tomography (micro-CT) and histology analyses in 4-or 8 week-old SAMP1/kl -/- mice. SAMP1/kl -/- mice exhibited extensive inflammation, tissue calcification, and abnormal mandibular bone development. Using micro-CT analysis, SAMP1/kl -/- mice displayed a loss of incisor roots and irregular dentinal tubule formation, as well as calcification within the pulp root canal. Furthermore, the mandibular ramus showed extensive ground glass appearance in SAMP1/kl -/- mice. In histological analysis, we found calcified skeletal structures and dysplastic bone formation in SAMP1/kl -/- mice. These results provide an understanding of the pathologic alterations of aging-related mandibular bone. SAMP1/kl -/- mice may serve as a novel model for dysosteogenesis in mandibular bone development.

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

Klotho (KL) is a single transmembrane protein composed of KL1 and KL2 repeats possessing β-glucuronidase activity and maintains calcium homeostasis in physiological state. It has been implicated in pigs that calcium is important for the establishment and maintenance of pregnancy, and our previous study has shown that transient receptor potential vanilloid type 6 (TRPV6), a calcium ion transporter, is predominantly expressed in the uterine endometrium during pregnancy in pigs. However, expression and function of KL in the uterine endometrium has not been determined in pigs. Thus, the present study determined expression and regulation of KL in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of KL mRNA decreased between Days 12 to 15 of the estrous cycle, and its expression showed a biphasic manner during pregnancy. KL mRNA was expressed in conceptuses and in chorioallantoic tissues during pregnancy. Explant culture study showed that expression levels of KL were not affected by treatment of steroid hormones or interleukin-1beta during the implantation period. Furthermore, levels of KL mRNA in the uterine endometrium from gilts carrying somatic cell nuclear transfer (SCNT)- derived embryos were significantly lower than those from gilts carrying natural mating-derived embryos on Day 12 of pregnancy. These results exhibited that KL was expressed at the maternal-conceptus interface in a pregnancy statusand stage-specific manner, and its expression was affected by SCNT procedure, suggesting that KL may play an important role in the establishment and maintenance of pregnancy in pigs.