Establishment of Transgenic Porcine Fibroblasts Expressing a Human klotho Gene and Its Effects on Gene Expression and Preimplantation Development of Cloned Embryos.

Even though the functions of the klotho gene in aging of small animals such as mice have been well investigated, 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. Therefore, the objective of this study was to generate porcine cell lines overexpressing human klotho (hKlotho) and tetracycline (Tet)-inducible hKlotho and to produce cloned embryos from these cell lines. We designed vectors for hKlotho overexpression (CA-Klotho) under control of CMV enhancer/chicken β-actin (CAG) promoter and Tet-inducible hKlotho overexpression (Tet-Klotho, under control of doxycycline-dependent promoter). The vectors were transfected into porcine fibroblasts then CA-Klotho and Tet-Klotho cell lines were established. The Tet-Klotho (+) cell line was cultured in the presence of doxycycline (2 μg/mL), whereas the Tet-Klotho (-) cell line was cultured without doxycycline. In polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) assays, integration and expression of the hKlotho gene were confirmed in CA-Klotho, Tet-Klotho (+), and Tet-Klotho (-) cell lines. The CA-Klotho cell line was subjected to real-time PCR and showed positively changed expression of genes related to aging and cell survival. Somatic cell nuclear transfer was performed to generate hKlotho overexpression cloned embryos by using CA-Klotho and Tet-Klotho (+) cell lines; blastocyst formation frequency was significantly higher in cloned embryos from CA-Klotho and Tet-Klotho (+) (21.5% and 20.2%, respectively) compared with the control (8.4%). In conclusion, we established hKlotho overexpression and Tet-inducible hKlotho overexpression cell lines and porcine embryos cloned from these cell lines.