Development of stable cell lines expressing high levels of point mutants of human opsin for biochemical and biophysical studies.
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 of the same opsin transgene linked to a different drug selection marker (hygro(R)) yielded expression clones with increased opsin levels compared to the neo(R) parent strain. Wild-type and mutant human opsins regenerate with native chromophore and demonstrate spectroscopic properties consistent with previous reports of bovine opsin mutants. HEK293S cells can be grown in larger scale suspension culture (10(9) cells/liter) or in roller bottles (10(8) cells/bottle) to facilitate milligram-order preparations of purified pigments. These cell lines should be useful in any time-resolved spectroscopic or biophysical experiments that require either uniform cellular levels of opsin protein or regenerable pigment, or large amounts of purified visual pigment. They should also be useful in experiments where uniform constitutive levels of a given mutant human visual pigment are needed in each cell. These and similar types of constitutive or inducible cell lines may also be useful for studying mechanisms of human cell death that occur by mutations in the human rod opsin gene.
Sullivan
J M
JM
Department of Ophthalmology, State University of New York Health Science Center, Syracuse 13210, USA.
Satchwell
M F
MF
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013053:Spectrophotometry; D014162:Transfection / Q000379:methods*
D017931:DNA Primers; D011994:Recombinant Proteins; D017299:Rod Opsins