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T.E. Liggett, E.R. Gaillard; Production of a Plasmid for the Stable Transfection of the Human ABCA–4 Gene Into a Mammalian Cell Line . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1706.
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Purpose: ABCA–4 is an ATP–binding cassette transport protein that is responsible for the flipping of all–trans retinal from the intramembrane space to the cytosolic space in the rod outer segments. Mutations within this gene have been suggested to play a role in Stargardt’s disease and Age Related Macular Degeneration. These diseases are characterized by a large accumulation of lipofuscin in the retinal pigment epithelium. It has been postulated that this accumulation is caused by improper functioning of the ABCA–4 protein. The purpose of this study is to produce a plasmid containing the human ABCA–4 gene that can be used for the stable transfection of a mammalian cell line. A stable transfection is preferred because it will allow the isolation of a clone to determine the protein’s in–vivo and in–vitro functional characteristics. The mammalian cell line should provide for the correct post–translational modifications. Methods: A full length ABCR gene was isolated from the pRK5 vector donated by Dr. Jeremy Nathans and incorporated into a plasmid as a fusion protein with markers for selectivity within mammalian cells and a 6X His tag. The ABCR gene was digested and amplified using PCR. The cDNA was ligated into the new plasmid and transformed in XL 10–Gold ultracompetent E. coli cells and then isolated. The DNA was extracted and analyzed by gel electrophoresis and partially sequenced and aligned. Results: The digestion of the pRK5 cells yielded the expected DNA size on gel electrophoresis. Analysis of the PCR amplification showed that the new ABCR gene was also of correct size. After ligation and transformation utilizing the new plasmid, it was run on a DNA agarose gel with both single and double digestions and plasmid was determined to contain the gene. The gene was isolated and sequenced analyzed. Conclusions: These results suggest that the ABCR gene was successfully incorporated into the plasmid. Current studies are underway to transfect a mammalian cell line and determine the cellular location of the expressed protein by confocal microscopy.
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