[3] Development of stable cell lines expressing high levels of point mutants of human opsin for biochemical and biophysical studies

摘要: Stable HEK293S cell lines expressing high levels of normal and mutant human rod opsins were generated. Cellular expression is uniform across a population. Secondary overexpression the same opsin transgene linked to different drug selection marker (hygro(R)) yielded clones with increased compared neo(R) parent strain. Wild-type regenerate native chromophore demonstrate spectroscopic properties consistent previous reports bovine mutants. cells can be grown in larger scale suspension culture (10(9) cells/liter) or roller bottles (10(8) cells/bottle) facilitate milligram-order preparations purified pigments. These should useful any time-resolved biophysical experiments that require either cellular protein regenerable pigment, large amounts visual pigment. They also where constitutive given pigment are needed each cell. similar types inducible may for studying mechanisms death occur by mutations gene.