| The klotho mouse is a unique model for human aging. A functional knock-out of the klotho gene yields a mouse that develops normally until puberty, then displays various phenotypes associated with human aging such as arteriosclerosis, osteoporosis, genital atrophy and reduced life span. Understanding klotho regulation and function promises a greater understanding of the human aging process as a whole.; To attempt a reversal of the aging phenotype, we designed lentiviral vectors capable of expressing the secreted form of klotho in cells. We also generated 293 clones expressing human klotho and a human klotho-GFP fusion protein to use in the dual membrane implantation device TheraCyte. For detection, three polyclonal antibodies were produced against murine klotho.; We then studied the regulation of klotho by luciferase assays. Initially, several cell lines were analyzed for klotho expression. 3T3, 293A, TCMK-1, MDCK, OK, and Hela cells were found negative by Northern. The RT-PCR being a more sensitive assay gave contradicting results with some positive cell lines. Real time PCR, with a probe and primer against murine klotho, confirmed that 3T3 and TCMK-1 cells were negative. In the absence of an endogenously expressing cell line, cells were chosen for luciferase assays according to the individual assay requirement. Interesting motifs were found upstream from exon 1, similarly in the mouse and human promoters. The klotho gene contains phosphate response element (PRE) motifs. The presence or absence of PRE under low or normal Pi conditions did not impart an enhancement of expression. The addition of the phosphate responsive transcription factor TFE3 did not change expression from the klotho promoters. HFH-3, a transcription factor expressed exclusively in the kidney and distal tubules, did not enhance expression from any of the klotho fragments despite the presence of its 13bps binding site. Long CT repeats in the deleted region of the klotho promoter were also studied, but no difference in expression was observed probably because they are proposed to work on higher order DNA structure and require genomic integration. |
