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Kyaw Soe, Marilyn Chwa, Khoa Pham, Nitin Udar, S M. Jazwinski, Michael Miceli, M C. Kenney; Developing Human ARPE-19 Cybrid Cell Lines from Different Haplogroups Related to Age-related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2011;52(14):2330.
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© ARVO (1962-2015); The Authors (2016-present)
AMD is a leading cause of vision loss in people over 65 years of age. Human populations can be classified based on their mitochondrial haplogroups defined by sets of single nucleotide polymorphisms (SNPs) within the mitochondrial DNA (mtDNA). Previous studies have shown that the mtDNA haplogroup J is associated with AMD while haplogroup H is not. The present study was designed to examine the influence of specific mtDNA haplogroups on mitochondrial functions by creating cytoplasmic hybrids (cybrids) which have identical nuclear genomes but differ only in the mitochondria.
ARPE-19 cells were depleted of their mitochondria (rho0 ) by successive passaging in a low concentrations ethidium bromide and supplementation with uridine. The mitochondrial depletion was confirmed by polymerase chain reaction (PCR) for nuclear DNA (18s) and mtDNA (cytochrome B, MT-CYB). Cybrids were created by using polyethylene glycol solution to fuse platelets from individuals with either H or J mtDNA haplogroups with rho0 ARPE-19 cells. Three to five days after fusion, media which lacked uridine was used to select for the fused ARPE-19 cybrids. Confirmation of cybrids was determined by PCR for the mtDNA encoded MT-CYB and J haplogroup (G13708A -1210bp).
By phase contrast microscopy, the cybrids appeared similar to rho0 ARPE-19 cells. The 18s band was present in ARPE-19 wild-type, rho0 ARPE-19 cells and the cybrids. The MT-CYB gene was present in the wild-type ARPE-19 and cybrids (H and J) but was lacking in the rho0 ARPE-19. The J haplogroup band at 1210 bp was present only in the J cybrids and absent in rho0 ARPE-19 cultures due to lack of mtDNA.
These findings demonstrate successful creation of human ARPE-19 cybrids with the SNP pattern that defines the J haplogroup. These cybrids are a novel tissue culture model to study the unique functions of specific mtDNA haplogroups.
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