Purpose. Rat retinal arrestin/S-Antigen (S-Ag) is one of several ocular autoantigens that, upon immunization of a susceptible host, can induce experimental autoimmune uveoretinitis (EAU). We created several eukaryotic cell lines that express high levels of wild type and mutant rat S-Ag. This was done so that S-Ag, using retroviral mediated gene transfer, could be expressed in vivo as an extraocular neo-self antigen in rats for the study of immunological tolerance. Methods. The S-Ag sequence was cloned into the retroviral vector pLXSN and the recombinant plasmid designated pS-Ag. A recombinant retroviral vector containing a mutated S-Ag sequence was made by inverse PCR on pS-Ag. The plasmid containing the mutated S-Ag was designated pS-AgΔ273 and carries a change in the amino acid sequence of the immunodominant epitope 273-289 (STLTKTLVLPLLANNR changed to STLTKRSNNR) to eliminate its immunological activity. The plasmids were transfected into the retroviral packaging cell line GP+E86 and the cells were selected in the antibiotic G418. These cells, designated GP-SAg, package S-Ag into an infectious but replication-deficient retrovirus. This virus was used to create two other cell lines that do not produce virus: 1) An anchorage independent T cell hybridoma line (KZO) was co-cultured with GP-SAg cells to create KZO-SAg cells and 2) NIH3T3 cells were infected with GP-SAg supernatant to create NIH-SAg cells. Rat S-Ag expression was assayed by western blotting. Results. All three cell lines express wild type or mutant rat S-Ag. The level of S-Ag expression could be enhanced by stimulating the cells with PMA and ionomycin. Conclusion. We have created several cells line that express high levels of wild type or mutant S-Ag that can serve as a source of S-Ag for in vitro studies. The quantity and ease of obtaining S-Ag from these cells is much greater than isolation from rat retina or from in vitro translation. The infectious nature of the retrovirus allows synthesis of rat S-Ag in a wide variety of rodent cells.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|